5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.","critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .","The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .","Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .",". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.","The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.","Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .","HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.","HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.","Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .","β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .","U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .","For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.","Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.","The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.","At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.","Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .","Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .","All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.","Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .","Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.","For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.","Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .","Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .","Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .","The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.","Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.","The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .","As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .","The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.","We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.","These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.",", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.",". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.","The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.","In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .","Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .",". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .","Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.",". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .","Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.","However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .",". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.",". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.",". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.","We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .",". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.","In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .","Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.","In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper."],"string":"[\n \"Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.\",\n \"In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .\",\n \"Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .\",\n \"The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .\",\n \". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .\",\n \". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .\",\n \"Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.\",\n \"We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.\",\n \"All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .\",\n \"The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .\",\n \": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.\",\n \"critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .\",\n \"The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .\",\n \"Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .\",\n \". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.\",\n \"The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.\",\n \"Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .\",\n \"HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.\",\n \"HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.\",\n \"Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .\",\n \"β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .\",\n \"U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .\",\n \"For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.\",\n \"Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.\",\n \"The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.\",\n \"At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.\",\n \"Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .\",\n \"Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .\",\n \"All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.\",\n \"Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \\\"Material and Methods. \\\" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .\",\n \"Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \\\"Material and Methods. \\\" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.\",\n \"For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.\",\n \"Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .\",\n \"Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .\",\n \"Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .\",\n \"The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.\",\n \"Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.\",\n \"The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \\\"Materials and Methods.\\\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .\",\n \"As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \\\"Materials and Methods.\\\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .\",\n \"The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.\",\n \"We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.\",\n \"These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.\",\n \", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.\",\n \". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.\",\n \"The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.\",\n \"In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .\",\n \"Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .\",\n \". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .\",\n \"Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.\",\n \". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .\",\n \"Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.\",\n \"However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .\",\n \". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.\",\n \". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.\",\n \". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.\",\n \"We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .\",\n \". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.\",\n \"In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .\",\n \"Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.\",\n \"In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper.\"\n]"},"document_id":{"kind":"number","value":2565,"string":"2,565"},"id":{"kind":"number","value":546,"string":"546"}}},{"rowIdx":662,"cells":{"question":{"kind":"string","value":"What is the structure of Hantaan virus?"},"answer":{"kind":"string","value":"enveloped, negative-sense RNA virus"},"context_chunks":{"kind":"list like","value":["Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.","In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .","Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .","The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .",". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .",". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .","Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.","We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.","All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .","The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .",": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.","critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .","The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .","Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .",". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.","The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.","Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .","HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.","HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.","Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .","β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .","U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .","For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.","Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.","The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.","At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.","Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .","Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .","All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.","Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .","Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.","For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.","Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .","Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .","Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .","The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.","Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.","The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .","As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .","The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.","We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.","These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.",", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.",". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.","The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.","In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .","Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .",". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .","Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.",". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .","Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.","However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .",". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.",". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.",". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.","We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .",". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.","In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .","Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.","In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper."],"string":"[\n \"Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.\",\n \"In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .\",\n \"Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .\",\n \"The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .\",\n \". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .\",\n \". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .\",\n \"Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.\",\n \"We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.\",\n \"All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .\",\n \"The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .\",\n \": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.\",\n \"critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .\",\n \"The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .\",\n \"Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .\",\n \". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.\",\n \"The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.\",\n \"Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .\",\n \"HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.\",\n \"HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.\",\n \"Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .\",\n \"β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .\",\n \"U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .\",\n \"For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.\",\n \"Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.\",\n \"The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.\",\n \"At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.\",\n \"Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .\",\n \"Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .\",\n \"All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.\",\n \"Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \\\"Material and Methods. \\\" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .\",\n \"Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \\\"Material and Methods. \\\" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.\",\n \"For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.\",\n \"Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .\",\n \"Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .\",\n \"Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .\",\n \"The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.\",\n \"Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.\",\n \"The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \\\"Materials and Methods.\\\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .\",\n \"As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \\\"Materials and Methods.\\\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .\",\n \"The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.\",\n \"We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.\",\n \"These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.\",\n \", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.\",\n \". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.\",\n \"The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.\",\n \"In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .\",\n \"Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .\",\n \". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .\",\n \"Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.\",\n \". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .\",\n \"Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.\",\n \"However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .\",\n \". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.\",\n \". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.\",\n \". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.\",\n \"We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .\",\n \". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.\",\n \"In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .\",\n \"Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.\",\n \"In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper.\"\n]"},"document_id":{"kind":"number","value":2565,"string":"2,565"},"id":{"kind":"number","value":547,"string":"547"}}},{"rowIdx":663,"cells":{"question":{"kind":"string","value":"What the animal vector reservoir for Hantaan virus?"},"answer":{"kind":"string","value":"rodents"},"context_chunks":{"kind":"list like","value":["Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.","In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .","Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .","The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .",". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .",". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .","Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.","We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.","All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .","The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .",": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.","critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .","The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .","Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .",". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.","The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.","Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .","HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.","HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.","Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .","β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .","U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .","For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.","Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.","The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.","At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.","Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .","Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .","All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.","Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .","Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.","For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.","Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .","Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .","Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .","The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.","Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.","The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .","As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .","The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.","We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.","These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.",", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.",". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.","The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.","In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .","Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .",". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .","Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.",". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .","Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.","However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .",". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.",". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.",". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.","We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .",". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.","In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .","Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.","In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper."],"string":"[\n \"Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.\",\n \"In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .\",\n \"Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .\",\n \"The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .\",\n \". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .\",\n \". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .\",\n \"Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.\",\n \"We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.\",\n \"All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .\",\n \"The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .\",\n \": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.\",\n \"critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .\",\n \"The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .\",\n \"Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .\",\n \". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.\",\n \"The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.\",\n \"Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .\",\n \"HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.\",\n \"HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.\",\n \"Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .\",\n \"β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .\",\n \"U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .\",\n \"For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.\",\n \"Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.\",\n \"The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.\",\n \"At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.\",\n \"Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .\",\n \"Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .\",\n \"All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.\",\n \"Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \\\"Material and Methods. \\\" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .\",\n \"Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \\\"Material and Methods. \\\" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.\",\n \"For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.\",\n \"Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .\",\n \"Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .\",\n \"Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .\",\n \"The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.\",\n \"Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.\",\n \"The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \\\"Materials and Methods.\\\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .\",\n \"As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \\\"Materials and Methods.\\\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .\",\n \"The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.\",\n \"We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.\",\n \"These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.\",\n \", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.\",\n \". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.\",\n \"The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.\",\n \"In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .\",\n \"Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .\",\n \". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .\",\n \"Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.\",\n \". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .\",\n \"Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.\",\n \"However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .\",\n \". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.\",\n \". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.\",\n \". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.\",\n \"We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .\",\n \". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.\",\n \"In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .\",\n \"Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.\",\n \"In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper.\"\n]"},"document_id":{"kind":"number","value":2565,"string":"2,565"},"id":{"kind":"number","value":548,"string":"548"}}},{"rowIdx":664,"cells":{"question":{"kind":"string","value":"What diagnostic test is correlated with the severity of HFRS?"},"answer":{"kind":"string","value":"plasma viral load"},"context_chunks":{"kind":"list like","value":["Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.","In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .","Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .","The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .",". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .",". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .","Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.","We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.","All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .","The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .",": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.","critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .","The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .","Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .",". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.","The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.","Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .","HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.","HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.","Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .","β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .","U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .","For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.","Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.","The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.","At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.","Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .","Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .","All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.","Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .","Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.","For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.","Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .","Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .","Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .","The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.","Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.","The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .","As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .","The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.","We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.","These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.",", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.",". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.","The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.","In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .","Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .",". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .","Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.",". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .","Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.","However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .",". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.",". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.",". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.","We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .",". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.","In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .","Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.","In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper."],"string":"[\n \"Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.\",\n \"In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .\",\n \"Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .\",\n \"The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .\",\n \". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .\",\n \". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .\",\n \"Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.\",\n \"We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.\",\n \"All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .\",\n \"The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .\",\n \": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.\",\n \"critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .\",\n \"The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .\",\n \"Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .\",\n \". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.\",\n \"The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.\",\n \"Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .\",\n \"HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.\",\n \"HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.\",\n \"Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .\",\n \"β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .\",\n \"U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .\",\n \"For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.\",\n \"Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.\",\n \"The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.\",\n \"At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.\",\n \"Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .\",\n \"Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .\",\n \"All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.\",\n \"Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \\\"Material and Methods. \\\" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .\",\n \"Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \\\"Material and Methods. \\\" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.\",\n \"For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.\",\n \"Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .\",\n \"Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .\",\n \"Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .\",\n \"The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.\",\n \"Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.\",\n \"The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \\\"Materials and Methods.\\\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .\",\n \"As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \\\"Materials and Methods.\\\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .\",\n \"The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.\",\n \"We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.\",\n \"These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.\",\n \", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.\",\n \". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.\",\n \"The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.\",\n \"In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .\",\n \"Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .\",\n \". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .\",\n \"Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.\",\n \". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .\",\n \"Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.\",\n \"However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .\",\n \". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.\",\n \". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.\",\n \". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.\",\n \"We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .\",\n \". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.\",\n \"In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .\",\n \"Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.\",\n \"In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper.\"\n]"},"document_id":{"kind":"number","value":2565,"string":"2,565"},"id":{"kind":"number","value":549,"string":"549"}}},{"rowIdx":665,"cells":{"question":{"kind":"string","value":"How is Interferon used in practice?"},"answer":{"kind":"string","value":"an antiviral medicine"},"context_chunks":{"kind":"list like","value":["Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.","In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .","Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .","The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .",". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .",". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .","Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.","We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.","All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .","The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .",": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.","critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .","The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .","Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .",". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.","The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.","Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .","HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.","HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.","Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .","β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .","U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .","For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.","Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.","The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.","At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.","Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .","Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .","All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.","Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .","Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.","For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.","Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .","Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .","Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .","The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.","Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.","The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .","As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .","The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.","We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.","These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.",", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.",". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.","The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.","In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .","Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .",". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .","Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.",". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .","Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.","However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .",". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.",". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.",". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.","We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .",". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.","In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .","Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.","In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper."],"string":"[\n \"Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.\",\n \"In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .\",\n \"Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .\",\n \"The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .\",\n \". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .\",\n \". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .\",\n \"Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.\",\n \"We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.\",\n \"All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .\",\n \"The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .\",\n \": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.\",\n \"critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .\",\n \"The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .\",\n \"Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .\",\n \". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.\",\n \"The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.\",\n \"Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .\",\n \"HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.\",\n \"HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.\",\n \"Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .\",\n \"β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .\",\n \"U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .\",\n \"For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.\",\n \"Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.\",\n \"The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.\",\n \"At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.\",\n \"Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .\",\n \"Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .\",\n \"All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.\",\n \"Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \\\"Material and Methods. \\\" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .\",\n \"Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \\\"Material and Methods. \\\" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.\",\n \"For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.\",\n \"Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .\",\n \"Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .\",\n \"Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .\",\n \"The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.\",\n \"Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.\",\n \"The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \\\"Materials and Methods.\\\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .\",\n \"As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \\\"Materials and Methods.\\\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .\",\n \"The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.\",\n \"We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.\",\n \"These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.\",\n \", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.\",\n \". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.\",\n \"The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.\",\n \"In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .\",\n \"Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .\",\n \". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .\",\n \"Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.\",\n \". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .\",\n \"Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.\",\n \"However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .\",\n \". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.\",\n \". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.\",\n \". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.\",\n \"We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .\",\n \". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.\",\n \"In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .\",\n \"Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.\",\n \"In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper.\"\n]"},"document_id":{"kind":"number","value":2565,"string":"2,565"},"id":{"kind":"number","value":551,"string":"551"}}},{"rowIdx":666,"cells":{"question":{"kind":"string","value":"What genotypes are associated with the severity of HFRS?"},"answer":{"kind":"string","value":"rs12252 C allele and CC genotype"},"context_chunks":{"kind":"list like","value":["Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.","In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .","Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .","The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .",". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .",". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .","Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.","We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.","All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .","The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .",": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.","critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .","The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .","Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .",". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.","The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.","Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .","HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.","HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.","Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .","β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .","U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .","For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.","Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.","The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.","At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.","Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .","Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .","All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.","Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .","Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.","For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.","Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .","Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .","Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .","The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.","Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.","The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .","As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .","The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.","We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.","These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.",", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.",". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.","The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.","In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .","Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .",". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .","Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.",". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .","Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.","However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .",". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.",". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.",". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.","We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .",". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.","In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .","Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.","In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper."],"string":"[\n \"Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.\",\n \"In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .\",\n \"Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .\",\n \"The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .\",\n \". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .\",\n \". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .\",\n \"Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.\",\n \"We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.\",\n \"All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .\",\n \"The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .\",\n \": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.\",\n \"critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .\",\n \"The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .\",\n \"Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .\",\n \". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.\",\n \"The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.\",\n \"Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .\",\n \"HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.\",\n \"HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.\",\n \"Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .\",\n \"β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .\",\n \"U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .\",\n \"For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.\",\n \"Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.\",\n \"The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.\",\n \"At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.\",\n \"Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .\",\n \"Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .\",\n \"All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.\",\n \"Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \\\"Material and Methods. \\\" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .\",\n \"Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \\\"Material and Methods. \\\" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.\",\n \"For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.\",\n \"Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .\",\n \"Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .\",\n \"Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .\",\n \"The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.\",\n \"Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.\",\n \"The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \\\"Materials and Methods.\\\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .\",\n \"As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \\\"Materials and Methods.\\\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .\",\n \"The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.\",\n \"We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.\",\n \"These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.\",\n \", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.\",\n \". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.\",\n \"The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.\",\n \"In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .\",\n \"Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .\",\n \". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .\",\n \"Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.\",\n \". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .\",\n \"Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.\",\n \"However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .\",\n \". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.\",\n \". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.\",\n \". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.\",\n \"We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .\",\n \". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.\",\n \"In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .\",\n \"Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.\",\n \"In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper.\"\n]"},"document_id":{"kind":"number","value":2565,"string":"2,565"},"id":{"kind":"number","value":552,"string":"552"}}},{"rowIdx":667,"cells":{"question":{"kind":"string","value":"Which IFITM proteins have been shown to possess antiviral activity?"},"answer":{"kind":"string","value":"IFITM1, 2, and 3"},"context_chunks":{"kind":"list like","value":["Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.","In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .","Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .","The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .",". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .",". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .","Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.","We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.","All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .","The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .",": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.","critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .","The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .","Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .",". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.","The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.","Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .","HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.","HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.","Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .","β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .","U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .","For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.","Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.","The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.","At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.","Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .","Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .","All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.","Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .","Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.","For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.","Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .","Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .","Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .","The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.","Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.","The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .","As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .","The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.","We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.","These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.",", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.",". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.","The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.","In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .","Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .",". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .","Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.",". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .","Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.","However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .",". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.",". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.",". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.","We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .",". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.","In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .","Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.","In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper."],"string":"[\n \"Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.\",\n \"In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .\",\n \"Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .\",\n \"The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .\",\n \". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .\",\n \". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .\",\n \"Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.\",\n \"We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.\",\n \"All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .\",\n \"The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .\",\n \": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.\",\n \"critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .\",\n \"The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .\",\n \"Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .\",\n \". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.\",\n \"The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.\",\n \"Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .\",\n \"HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.\",\n \"HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.\",\n \"Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .\",\n \"β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .\",\n \"U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .\",\n \"For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.\",\n \"Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.\",\n \"The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.\",\n \"At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.\",\n \"Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .\",\n \"Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .\",\n \"All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.\",\n \"Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \\\"Material and Methods. \\\" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .\",\n \"Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \\\"Material and Methods. \\\" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.\",\n \"For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.\",\n \"Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .\",\n \"Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .\",\n \"Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .\",\n \"The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.\",\n \"Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.\",\n \"The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \\\"Materials and Methods.\\\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .\",\n \"As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \\\"Materials and Methods.\\\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .\",\n \"The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.\",\n \"We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.\",\n \"These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.\",\n \", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.\",\n \". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.\",\n \"The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.\",\n \"In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .\",\n \"Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .\",\n \". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .\",\n \"Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.\",\n \". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .\",\n \"Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.\",\n \"However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .\",\n \". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.\",\n \". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.\",\n \". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.\",\n \"We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .\",\n \". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.\",\n \"In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .\",\n \"Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.\",\n \"In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper.\"\n]"},"document_id":{"kind":"number","value":2565,"string":"2,565"},"id":{"kind":"number","value":554,"string":"554"}}},{"rowIdx":668,"cells":{"question":{"kind":"string","value":"What is the hypothesized mechanism by which IFITMs work?"},"answer":{"kind":"string","value":"restrict viral infection at the stage of cellular entry"},"context_chunks":{"kind":"list like","value":["Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.","In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .","Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .","The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .",". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .",". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .","Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.","We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.","All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .","The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .",": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.","critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .","The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .","Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .",". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.","The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.","Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .","HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.","HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.","Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .","β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .","U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .","For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.","Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.","The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.","At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.","Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .","Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .","All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.","Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .","Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.","For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.","Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .","Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .","Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .","The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.","Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.","The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .","As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .","The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.","We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.","These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.",", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.",". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.","The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.","In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .","Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .",". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .","Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.",". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .","Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.","However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .",". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.",". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.",". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.","We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .",". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.","In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .","Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.","In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper."],"string":"[\n \"Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.\",\n \"In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .\",\n \"Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .\",\n \"The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .\",\n \". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .\",\n \". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .\",\n \"Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.\",\n \"We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.\",\n \"All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .\",\n \"The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .\",\n \": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.\",\n \"critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .\",\n \"The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .\",\n \"Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .\",\n \". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.\",\n \"The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.\",\n \"Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .\",\n \"HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.\",\n \"HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.\",\n \"Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .\",\n \"β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .\",\n \"U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .\",\n \"For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.\",\n \"Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.\",\n \"The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.\",\n \"At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.\",\n \"Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .\",\n \"Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .\",\n \"All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.\",\n \"Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \\\"Material and Methods. \\\" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .\",\n \"Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \\\"Material and Methods. \\\" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.\",\n \"For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.\",\n \"Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .\",\n \"Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .\",\n \"Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .\",\n \"The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.\",\n \"Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.\",\n \"The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \\\"Materials and Methods.\\\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .\",\n \"As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \\\"Materials and Methods.\\\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .\",\n \"The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.\",\n \"We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.\",\n \"These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.\",\n \", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.\",\n \". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.\",\n \"The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.\",\n \"In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .\",\n \"Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .\",\n \". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .\",\n \"Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.\",\n \". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .\",\n \"Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.\",\n \"However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .\",\n \". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.\",\n \". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.\",\n \". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.\",\n \"We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .\",\n \". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.\",\n \"In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .\",\n \"Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.\",\n \"In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper.\"\n]"},"document_id":{"kind":"number","value":2565,"string":"2,565"},"id":{"kind":"number","value":555,"string":"555"}}},{"rowIdx":669,"cells":{"question":{"kind":"string","value":"What genotype causes truncation of the IFITM3 protein?"},"answer":{"kind":"string","value":"rs12252 C allele"},"context_chunks":{"kind":"list like","value":["Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.","In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .","Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .","The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .",". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .",". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .","Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.","We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.","All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .","The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .",": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.","critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .","The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .","Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .",". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.","The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.","Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .","HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.","HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.","Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .","β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .","U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .","For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.","Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.","The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.","At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.","Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .","Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .","All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.","Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .","Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.","For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.","Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .","Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .","Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .","The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.","Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.","The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .","As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .","The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.","We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.","These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.",", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.",". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.","The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.","In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .","Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .",". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .","Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.",". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .","Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.","However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .",". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.",". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.",". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.","We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .",". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.","In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .","Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.","In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper."],"string":"[\n \"Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.\",\n \"In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .\",\n \"Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .\",\n \"The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .\",\n \". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .\",\n \". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .\",\n \"Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.\",\n \"We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.\",\n \"All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .\",\n \"The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .\",\n \": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.\",\n \"critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .\",\n \"The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .\",\n \"Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .\",\n \". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.\",\n \"The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.\",\n \"Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .\",\n \"HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.\",\n \"HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.\",\n \"Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .\",\n \"β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .\",\n \"U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .\",\n \"For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.\",\n \"Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.\",\n \"The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.\",\n \"At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.\",\n \"Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .\",\n \"Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .\",\n \"All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.\",\n \"Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \\\"Material and Methods. \\\" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .\",\n \"Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \\\"Material and Methods. \\\" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.\",\n \"For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.\",\n \"Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .\",\n \"Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .\",\n \"Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .\",\n \"The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.\",\n \"Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.\",\n \"The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \\\"Materials and Methods.\\\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .\",\n \"As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \\\"Materials and Methods.\\\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .\",\n \"The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.\",\n \"We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.\",\n \"These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.\",\n \", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.\",\n \". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.\",\n \"The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.\",\n \"In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .\",\n \"Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .\",\n \". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .\",\n \"Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.\",\n \". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .\",\n \"Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.\",\n \"However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .\",\n \". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.\",\n \". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.\",\n \". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.\",\n \"We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .\",\n \". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.\",\n \"In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .\",\n \"Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.\",\n \"In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper.\"\n]"},"document_id":{"kind":"number","value":2565,"string":"2,565"},"id":{"kind":"number","value":556,"string":"556"}}},{"rowIdx":670,"cells":{"question":{"kind":"string","value":"What is a Hantavirus?"},"answer":{"kind":"string","value":"zoonotic diseases"},"context_chunks":{"kind":"list like","value":["Hantavirus infection, which causes zoonotic diseases with a high mortality rate in humans, has long been a global public health concern. Over the past decades, accumulating evidence suggests that long noncoding RNAs lncRNAs play key regulatory roles in innate immunity. However, the involvement of host lncRNAs in hantaviral control remains uncharacterized. In this study, we identified the lncRNA NEAT1 as a vital antiviral modulator. NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection.","NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection. Further investigation suggested that NEAT1 served as positive feedback for RIG-I signaling. HTNV infection activated NEAT1 transcription through the RIG-I–IRF7 pathway, whereas NEAT1 removed the transcriptional inhibitory effects of the splicing factor proline- and glutamine-rich protein SFPQ by relocating SFPQ to paraspeckles, thus promoting the expression of RIG-I and DDX60. RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections.","RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections. IMPORTANCE Hantaviruses have attracted worldwide attention as archetypal emerging pathogens. Recently, increasing evidence has highlighted long noncoding RNAs lncRNAs as key regulators of innate immunity; however, their roles in hantavirus infection remain unknown. In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling.","In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling. This lncRNA was induced by HTNV through the RIG-I–IRF7 pathway in a time- and dose-dependent manner and promoted HTNV-induced IFN production by facilitating RIG-I and DDX60 expression. Intriguingly, NEAT1 relocated SFPQ and formed paraspeckles after HTNV infection, which might reverse inhibitive effects of SFPQ on the transcription of RIG-I and DDX60. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections.","To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections. Text: glycoprotein GP , and viral RNA-dependent polymerase protein RdRp , respectively. Humans become infected by inhaling contaminated aerosols or by coming into contact with rodent excreta, and they develop two severe acute diseases, namely, hemorrhagic fever with renal syndrome HFRS and hantavirus pulmonary syndrome HPS . . Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . .",". Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . . Chinese HFRS cases, mainly caused by Hantaan virus HTNV infection, account for approximately 90% of all global cases, with a mortality rate ranging from 0.1 to 15% . . Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions.","Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions. Cellular pathogen recognition receptors PRRs , including Toll-like receptors TLRs and RIG-I like receptors RLRs , can detect distinct pathogen-associated molecular patterns PAMPs and trigger the expression of IFNs and cytokines. RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression.","RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression. lncRNA nuclear paraspeckle assembly transcript 1 NEAT1 is an essential architectural constituent of paraspeckles in the mammalian nucleus, interacting with Drosophila DBHS RNA-binding proteins such as the splicing factor proline-and glutamine-rich protein SFPQ and the non-POU domain-containing, octamer-binding protein NONO/p54 . . To date, two isoform transcripts of the NEAT1 gene have been identified, namely, the 3.7-kb NEAT1-1 MEN and the 23-kb NEAT1-2 MEN Fig. 1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . .","1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . . , promoting cancer formation in mice by dampening oncogene-dependent activation of p53 . . Nevertheless, studies assessing the function of NEAT1 in viral infections are scarce. Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication.","Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication. Further investigation showed that NEAT1 promoted RIG-I and DDX60 expression by relocating SFPQ and removing the transcriptional inhibitory effects of SFPQ, which are critical for IFN responses against HTNV infection. We also found that RIG-I signaling, rather than TLR3 and TLR4, accounted for the elevation of HTNV-induced NEAT1. Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection.","Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection. As shown in Fig. 1B , the NEAT1 level in the HTNV group was higher than that in the mock group P ϭ 6.86 ϫ 10 Ϫ13 , false discovery rate FDR ϭ 9.75 ϫ 10 Ϫ12 or the 60 Co-inactivated HTNV group P ϭ 1.75 ϫ 10 Ϫ14 , FDR ϭ 3.10 ϫ 10 Ϫ13 ; however, the difference between the 60 Co-inactivated HTNV group and the mock group was not significant P ϭ 0.21034, FDR ϭ 0.58211 . To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig.","To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig. 1A , were applied to quantify NEAT1 RNA isoforms by quantitative real-time PCR qRT-PCR . It has been reported that NEAT1-2 rather than NEAT1-1 plays a key regulatory role in paraspeckle formation . , and we also found that elevated NEAT1 levels depend on live HTNV infection rather than 60 Co-inactivated HTNV stimulation Fig. 1C . Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D .","Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D . To further investigate whether NEAT1 expression was altered in other cell lines, HEK293, HeLa, and A549 cells were used. All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G .","All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G . Similar to the data obtained from HUVECs, NEAT1 was indeed upregulated by HTNV at a multiplicity of infection MOI of 1 beginning at 24 hpi in HUVECs and A549, HEK293, and HeLa cells, and the increasing tendency occurred in a time-dependent manner Fig. 1H . Of note, the NEAT1 elevation at 2 hpi might have been unrelated to the virus but resulted in cellular stress responses. Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I .","Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I . NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. The abovedescribed data showed that HTNV infection increased NEAT1, and we wondered how NEAT1 could reciprocally influence HTNV replication. The small interfering RNA siRNA transfection efficiency in HUVECs was confirmed by flow cytometry, and NEAT1 expression was significantly decreased, as assessed by qRT-PCR after RNA interference RNAi Fig. 2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2.","2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2. Compared with the cells transfected with control siRNA negative control NC , HUVECs with si-NEAT1 could dramatically promote HTNV NP production, and NP expression seemed to be related to the amount of applied si-NEAT1 Fig. 2B . Intriguingly, depletion of NEAT1-2 alone could mimic the antiviral effects of simultaneous NEAT1-1 and NEAT1-2 silencing Fig. 2C , indicating that NEAT1-2 was critical for the antiviral responses. Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing.","Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing. On the other hand, plasmids, of which pCMV-NEAT1-1 is transcribed into the 3.7-kb NEAT1-1 MEN and pCMV-NEAT1-2 is transcribed into the 2-to 3-kb NEAT1-2 MEN , were applied to directly investigate the role of NEAT1 in HTNV infection Fig. 2F . Surprisingly, we found NEAT1-1 overexpression restricted NEAT1-2 transcription Fig. 2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G .","2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G . Furthermore, overexpression of NEAT1-2 instead of NEAT1-1 could efficiently suppress HTNV replication Fig. 2H . NEAT1-1 upregulation even aggravated HTNV infection Fig. 2H , which may be the result of downregulation of NEAT1-2. Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig.","Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig. 2J were limited in HUVECs in which NEAT1-2 was ectopically expressed in comparison to those transfected with control vector or pCMV-NEAT1-1. These data further showed that NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. Alteration of NEAT1-2 affects HTNV-induced IFN expression in HUVECs. IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . .","IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . . It has been reported that the GnT of hantaviruses suppressed IFN- expression of host cells at an early stage of infection . . Here, we also found that HUVECs could not efficiently produce IFN- until 12 hpi at an MOI of 0.1 or until 24 hpi at an MOI of 1 Fig. 3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig.","3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig. 3B , suggesting that NEAT1-2 expression increased just before IFN production. We found that expression of endogenous IFN- mRNA was much lower in cells transfected with si-NEAT1-2 at MOIs of both 0.1 Fig. 3C and 1 Fig. 3D than in those transfected with control siRNA NC . In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E .","In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E . More importantly, HUVECs transfected with pCMV-NEAT1-2 conspicuously increased IFN- gene expression compared with those cells with vector plasmids at 12 hpi MOI ϭ 1 , demonstrating that NEAT1-2 overexpression accelerated robust IFN responses in host cells against HTNV infection. With a dual luciferase reporter system Twenty-four hours after transfection, the cells expressing FAM were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1.","Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the NC group . NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g .","NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 0.1 for 48 h. The expression of HTNV NP was measured by Western blotting. C HUVECs were treated as described for panel A, right, but at an MOI of 0.1. In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.","In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NC group . D HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group .","The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . E HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g .","Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g . Twenty-four hours after transfection, the cells expressing green fluorescent protein GFP were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with control plasmids vector , pCMV-NEAT1-1, or pCMV-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig.","Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig. 3F . These results showed that NEAT1-2 regulated HTNV-induced IFN- expression. To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig.","To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig. 3G . In addition, in cells with high NEAT1-2 expression, treatment with neutralizing antibodies NAbs of IFN-␣ and IFN- could counteract the antiviral effects of NEAT1-2 MOI ϭ 1 , and the compensatory effects were dependent on the magnitude of the NAbs. Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production.","Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production. PRRs maintain a vital role in the promotion of IFN responses, and we conjectured that NEAT1 might amplify IFN responses by modulating these molecules. TLR3, TLR4, and RIG-I have been shown to recognize HTNV infection . . DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate .",". DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate . , Here, we found that multiple Toll-like receptors like TLR1, TLR2, TLR3, and TLR4, as well as MDA5, were increased after HTNV infection, but none of them were influenced by silencing NEAT1-2 Fig. 4A . The upregulated RIG-I and DDX60 were blocked in the cells with low NEAT1-2 expression after HTNV infection Fig. 4A . HUVECs with declining NEAT1-2 expression showed gradually decreasing expression of RIG-I and DDX60 Fig. 4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C .","4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C . These data indicated that NEAT1-2 could positively modulate RIG-I and DDX60 expression, while the role of RIG-I and DDX60 upon HTNV infection is obscure. We then found that RIG-I and DDX60 colocalized after HTNV infection Fig. 4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown .","4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown . Simultaneously knocking down RIG-I and DDX60 significantly promoted HTNV NP expression Fig. 4E , and knockdown of both of them could greatly affect IFN- expression Fig. 4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig.","4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig. 4H , indicating that efficient anti-HTNV responses might depend on the interactive effects of DDX60 and RIG-I. More importantly, RIG-I or/and DDX60 overexpression enhanced HTNV-induced IFN- expression, and they had synergistic effects on IFN- production Fig. 4I and J . Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60.","Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60. NEAT1 was found to interact with SFPQ by RNA immunoprecipitation RIP after HTNV infection Fig. 5A , indicating that modulatory effects of NEAT1 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively .","Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1 for 48 h. The expression of HTNV NP was measured by Western blotting. H HUVECs were treated as described for panel F, right. In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.","In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . H HUVECs were treated as described for panel F, right. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group .","The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . I HUVECs were treated as described for panel F, right. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ.","Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ. Interestingly, the protein level of SFPQ, as well as another paraspeckle-forming constituent, NONO, remained unchanged after HTNV infection Fig. 5B or after NEAT1 overexpression and knockdown Fig. 5C . However, SFPQ became centralized rather than diffuse in the nucleus after HTNV infection Fig. 5D . The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig.","The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig. 5F and G , which might have been related to the increase in RIG-I Fig. 5H and DDX60 Fig. 5I . SFPQ has been suggested to bind to the promoter region of RIG-I and DDX60 ., thus preventing the expression of RIG-I and DDX60. Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription.","Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription. Interestingly, overexpression of the S or M segment of HTNV in HEK293 cells failed to induce NEAT1 expression, suggesting that NEAT1 transcription was closely related to live viral replication Fig. 6A . Of note, the upregulation of NEAT1 by HTNV could not be reversed by applying IFN-I neutralizing antibodies Fig. 6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig.","6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig. 6C, D, and E or cytokines Fig. 6F and G . We conjectured that NEAT1 expression was related to the activation of PRRs. By knocking down several PRRs, we found that the RIG-I and TLR4 pathways played important roles in HTNV-induced NEAT1 upregulation Fig. 7A . Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C .","Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C . Moreover, using STAT1 as a positive control, we found that the transcription factor IRF7, rather than IRF3 and p65, translocated into the nucleus in HTNV-infected HUVECs at 2 dpi Fig. 7D . Furthermore, IRF7 knockdown blocked HTNV-induced NEAT1 upregulation Fig. 7E . Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice.","Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice. Although cell-based experiments revealed that NEAT1-2 is a crucial regulator of innate antihantaviral responses, its function in vivo has remained unclear. To address this question, we intravenously injected siRNAs targeting mouse NEAT1-2 at 1 day before HTNV infection. NEAT1-2 expression levels in the liver, kidney, and spleen were reduced at 2 dpi Fig. 8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions.","8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions. Body weight loss in NEAT1-2-depleted mice was observed from 2 dpi to 5 dpi, and the IFN production in serum was remarkably decreased in the NEAT1-2 silenced group than those in the NC group at 3 dpi Fig. 8B . As expected, NEAT1-2 knockdown mice showed considerably higher HTNV NP levels in the liver, spleen, and kidney at 3 dpi Fig. 8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D .","8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D . In addition, reduced inflammatory cell filtration but increased tissue injury was found in NEAT1-2 knockdown mice during the early stage of infection Fig. 8E . Infiltration of macrophages in the spleen was attenuated Fig. 8F , and the activation of macrophages was also suppressed by flow cytometry; data not shown . Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G .","Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G . Nevertheless, NEAT1-2 silencing had no effect on the production of neutralizing antibodies at 7 dpi data not shown . The above-described findings indicated that NEAT1-2 depletion might influence multiple aspects of the innate immune response in HTNV-infected mice. Innate immunity is a phylogenetically ancient and conserved system that counteracts invading microbes, the regulatory mechanism of which is sophisticated and complex. Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . .","Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . . In this report, we first demonstrated that NEAT1 was induced by HTNV through the RIG-I-IRF7 pathway and served as positive feedback for RIG-I signaling. Using DGE analysis, we observed upregulated NEAT1 and confirmed its alteration in To further determine the function of NEAT1 after HTNV infection in vivo, mice were injected intravenously with si-NEAT1-2 1 g/g or nontarget control siRNA NC 1 g/g ; 1 day later, they were infected with HTNV 100 LD 50 by intramuscular injection. A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group .","A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group . B The effects of NEAT1 on HTNV virulence in mice were determined by body weight loss from 0 to 10 dpi left panel, n ϭ 10 in each group . The IFN- in sera of different groups was measured by ELISA at 3dpi right panel, n ϭ 8 in each group . Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi.","Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi. Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . D NEAT1 effects on HTNV infection kinetics at 3 dpi were determined by testing the HTNV titers in livers, spleens, and kidneys. Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group .","Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group . After HTNV infection, livers in the NC group showed inflammatory cell infiltration in certain regions, while those in the si-NEAT1-2 group showed slight acute viral hepatitis. Spleens in the NC group showed lymph node hyperplasia, while those in the si-NEAT1-2 group were severely congestive. Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented.","Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented. G CD3 ϩ CD8 ϩ IFN-␥ ϩ T cells were analyzed by flow cytometry at 3 dpi, and the results obtained for three mice in each group are presented. different cell lines. To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression.","To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression. By virtue of RNAi, the RIG-I-IRF7 pathway was confirmed to be necessary for HTNV-triggered NEAT1 elevation. Recently, large-scale transcriptomic studies identified numerous noncoding transcripts in the mammalian genome, which were speculated to influence diverse biological processes. Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression .","Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression . . TLR2 activation or tumor necrosis factor alpha TNF-␣ stimulation induces transcription of the lncRNA THRIL, the downregulation of which impairs TNF-␣ and IL-6 secretion . . TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . .",". TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . . The lncRNA Lethe, triggered by TNF-␣ and IL-1, acts as a negative feedback regulator of NF-B signaling . . The roles of lncRNAs in host-virus interactions have been progressively unveiled. Various viruses, such as influenza virus IAV , coronavirus, enterovirus, human immunodeficiency virus HIV , hepatitis B virus HBV , hepatitis C virus HCV , Japanese encephalitis virus JEV , and rabies virus, have been reported to activate the transcription of different lncRNAs in host cells 11, . . . .",". . . Importantly, multiple lncRNAs have been shown to affect the IFN response in recent years and have gradually become hot spots in the field of antiviral research. NeST was shown to enhance IFN-␥ production, controlling the susceptibility of mice to persistent Theiler's virus infection as well as resistance to Salmonella enterica serovar Typhimurium infection . . Both CMPK2 and NRAV were identified as negative regulators of IFN immune reactions. CMPK2, induced by IFN-␣ or HCV infection, suppresses various ISGs, the knockdown of which dramatically blocks HCV replication . . NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . .",". NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . . Numerous lncRNAs, including lnc-ISG15 and ISR2, respond to IFNs such as ISGs, although their actual function requires further investigation . . Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection.","Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection. NEAT1 has been reported to interact with Drosophila DBHS RNA-binding proteins e.g., SFPQ, NONO-p54nrb, and PSPC1 , recruiting them to paraspeckles, a nuclear substructure found in all cultured and primary cells except embryonic stem cells . . The versatile function of NEAT1 is rapidly progressing in multiple areas of biology. NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . .","NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . . NEAT1 also participates in neurodegenerative diseases such as Huntington's disease . and seems to potentially contribute to the elevated production of a number of cytokines and chemokines in patients with systemic lupus erythematosus SLE . . Furthermore, poly I·C can activate NEAT1 transcription through the TLR3 pathway, whereas NEAT1 positively regulates IL-8 transcription and potentially affects the expression of multiple ISGs after poly I·C stimulation . . In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . .","In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . . However, the role of NEAT1 in hantaviral infection remains unclear. In this report, NEAT1 has been identified as an important regulator of the host innate immune system against HTNV infection. Elevated NEAT1 promotes IFN secretion, most likely by enhancing RIG-I and DDX60 expression. DDX60, a DEXD/H box RNA helicase similar to Saccharomyces cerevisiae Ski2, is induced after viral infection . . DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response.",". DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response. However, the antiviral effects of DDX60 seem to vary among viruses . . We found that NEAT1-regulated DDX60 was involved in IFN production in response to HTNV infection. In HTNV-infected cells, double-stranded RNA dsRNA could not be detected, and it is unclear how host PRRs, especially RIG-I, recognize HTNV invasion . . Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation.",". Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation. Of note, we applied multiple cell lines to explore the role of NEAT1 during HTNV infection. HTNV primarily targets vascular endothelial cells in vivo and contributes to the increased vascular permeability and coagulation disorders in HFRS; hence, HUVECs are the most common in vitro cell model to study host innate immunity against HTNV infection or viral pathogenesis . . EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies .",". EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies . . A549 cells were once used to isolate HTNV, and they were confirmed to be a mature model of infection . . . . . Additionally, Huh 7.0 and Huh 7.5 RIG-I Ϫ cells used in our study have been reported to be infected by HTNV by Lee et al. . and can be used as a cell model to study immune responses against HTNV replication . . Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV.",". Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV. Using qRT-PCR, Western blotting, and immunofluorescence assays, we have also shown that both HEK293 and HeLa cells can be infected by HTNV. To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production.","To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production. Alterations in the relative fluorescence intensity of NP after silencing or overexpressing NEAT1-2 did not seem to be as remarkable as qRT-PCR or Western blot analysis results. The NP spotted and exhibited in the ICW results forms obvious stains that mimic PFU. However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays.","However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays. The RNAi studies in vivo are encouraging Fig. 8 , but the NC used by our group was not mutated si-NEAT1-2 i.e., same sense strand, but with a point mutation in the targeting strand . The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling.","The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling. After observing that NEAT1 can regulate IFN expression by HTNV infection, we were interested in the function of NEAT1. We noticed that silencing NEAT1-2 or ectopically expressing NEAT1-2 could not inhibit or enhance IFN expression without HTNV infection Fig. 3F , which indicated that NEAT1-2 could not directly affect IFN- expression. This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression.","This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression. Recent findings have shown that RIG-I signaling is essential for an efficient polyfunctional T cell response during IAV infection . . Indeed, we found that the function of T cells was suppressed after NEAT1-2 depletion in our animal experiments Fig. 8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection.","8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection. HTNV infection induced NEAT1 expression through the RIG-I-IRF7 pathway, while NEAT1 displayed positive feedback for RIG-I signaling. NEAT1 relocated SFPQ from the potential promoter region of several antiviral genes to the paraspeckles, removing the transcriptional inhibitory effects of SFPQ. This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 .","This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 . nontarget control i.e., negative control NC or targeted RIG-I and DDX60 were designed by Gene-Pharma as follows: NC, 5=- UUCUUCGAACGUGUCACGUTT-3=; si-RIG-I-1, 5=-GCCCAUUUAAACCAAGAAATT-3=, si-RIG-I-2, 5=-GGUGGAGGAUAUUUGAACUTT-3=, and si-RIG-I-3, 5=-CCCAACGAUAUCAUUUCUTT-3=; si-DDX60-1, 5=-GUCCAGGUGUCAGUUUGAUTT-3=, si-DDX60-2, 5=-CCGAAGUGAAGAAGGUAAATT-3=, and si-DDX60-3, 5=-GAUGGAUGCUAGGAAAUAUTT-3=. The pCMV-NEAT1-1 and pCMV-NEAT1-2 plasmids, which transcribe NEAT1-1 and NEAT1-2, respectively, were provided by Nakagawa Shinichi . . The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . .",". The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . . Abs against RIG-I, IRF3, IRF7, p65, and STAT1, as well as the neutralizing antibodies against IFN-␣ and IFN-, were purchased from Abcam Cambridge, MA, USA . Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China .","Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China . The Abs targeting CD11b, F4/80, CD3, CD8, IFN-␥, inducible nitric oxide synthase iNOS , and CD206 for flow cytometry were purchased from BD Biosciences San Jose, CA, USA . IFN-␣, -, and -␥, TNF-␣, and IL-1 were from PeperoTech Rocky Hill, NJ . ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis.","ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis. Total cellular RNAs were extracted with RNAiso TaKaRa, Dalian, China , the concentration of which was measured using a NanoDrop 1000 spectrophotometer. Reverse transcription RT was then performed with PrimeScript RT master mix TaKaRa according to the instructions provided by the manufacturer. Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed.","Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed. The qRT-PCR primer sequences for NEAT1, NEAT1-2, IFN-, HTNV S segment, RIG-I, DDX60, -actin, and GAPDH were obtained from previous reports . . The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing.",". The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing. HUVECs with a confluence of 80% in 6 wells were mock infected or infected with live or 60 Co-inactivated HTNV at an MOI of 1. RNAs were extracted as previously described at 24 hpi, and the quality was analyzed using FastQC software by the Beijing Genomics Institute BGI, Shenzhen, China . Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded.","Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded. Tag counts were normalized to TPM transcripts per million by dividing the raw tag count by the total number of tags from each library and multiplying by 1 million. To avoid the possible noise signal from high-throughput sequencing, the genes with average TPM of less than 1 in these three states were excluded. In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01.","In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01. Genes were selected as differentially expressed using a P value threshold of 0.01. FISH and immunofluorescence assays IFA . Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature.","Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature. Prehybridization was performed with lncRNA FISH probe mix at 37°C for 30 min, and then hybridization was performed by adding NEAT1-2 FISH probe mix and incubating the mixture at 37°C overnight. After washing with 4ϫ, 2ϫ, and 1ϫ SSC, the cell nuclei were stained with DAPI 4=,6-diamidino-2-phenylindole . Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently.","Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently. The cells were fixed with 4% PFA for 10 min and permeabilized with 0.1% Triton X-100 for 15 min. Primary Abs were added and incubated at 37°C for 2 h. After five washes with DPBS, secondary Cy3-or fluorescein isothiocyanate FITC -conjugated goat anti-rabbit or goat anti-mouse IgG Sangon, Shanghai, China was added and incubated at 37°C for 2 h. Cell nuclei were stained with DAPI. Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue .","Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue . The samples were then boiled at 95°C for 10 min. The lysates were resolved by 10%, 12%, or 15% SDS-PAGE and transferred to polyvinylidene fluoride PVDF membranes Millipore . The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA .","The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA . ICW assay. The In-Cell Western ICW assay was performed using an Odyssey imaging system Li-Cor according to the manufacturer's instructions. HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1.","HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1. At 48 hpi, HUVECs were washed twice with ice-cold DPBS, fixed with 4% PFA for 10 min, and permeabilized with 1.0% Triton X-100 for 15 min. Cells were added with Li-Cor Odyssey blocking solution at room temperature for 30 min and incubated at 4°C overnight with mouse IgG MAb 1A8 against HTNV NP together with rabbit IgG antibody against -actin, both of which were diluted in PBS containing 3% bovine serum albumin BSA; HyClone . Subsequently, the cells were washed and stained with goat anti-mouse IgG IRDye 800 antibody 1:5,000; Li-Cor and goat anti-rabbit"],"string":"[\n \"Hantavirus infection, which causes zoonotic diseases with a high mortality rate in humans, has long been a global public health concern. Over the past decades, accumulating evidence suggests that long noncoding RNAs lncRNAs play key regulatory roles in innate immunity. However, the involvement of host lncRNAs in hantaviral control remains uncharacterized. In this study, we identified the lncRNA NEAT1 as a vital antiviral modulator. NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection.\",\n \"NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection. Further investigation suggested that NEAT1 served as positive feedback for RIG-I signaling. HTNV infection activated NEAT1 transcription through the RIG-I–IRF7 pathway, whereas NEAT1 removed the transcriptional inhibitory effects of the splicing factor proline- and glutamine-rich protein SFPQ by relocating SFPQ to paraspeckles, thus promoting the expression of RIG-I and DDX60. RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections.\",\n \"RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections. IMPORTANCE Hantaviruses have attracted worldwide attention as archetypal emerging pathogens. Recently, increasing evidence has highlighted long noncoding RNAs lncRNAs as key regulators of innate immunity; however, their roles in hantavirus infection remain unknown. In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling.\",\n \"In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling. This lncRNA was induced by HTNV through the RIG-I–IRF7 pathway in a time- and dose-dependent manner and promoted HTNV-induced IFN production by facilitating RIG-I and DDX60 expression. Intriguingly, NEAT1 relocated SFPQ and formed paraspeckles after HTNV infection, which might reverse inhibitive effects of SFPQ on the transcription of RIG-I and DDX60. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections.\",\n \"To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections. Text: glycoprotein GP , and viral RNA-dependent polymerase protein RdRp , respectively. Humans become infected by inhaling contaminated aerosols or by coming into contact with rodent excreta, and they develop two severe acute diseases, namely, hemorrhagic fever with renal syndrome HFRS and hantavirus pulmonary syndrome HPS . . Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . .\",\n \". Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . . Chinese HFRS cases, mainly caused by Hantaan virus HTNV infection, account for approximately 90% of all global cases, with a mortality rate ranging from 0.1 to 15% . . Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions.\",\n \"Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions. Cellular pathogen recognition receptors PRRs , including Toll-like receptors TLRs and RIG-I like receptors RLRs , can detect distinct pathogen-associated molecular patterns PAMPs and trigger the expression of IFNs and cytokines. RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression.\",\n \"RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression. lncRNA nuclear paraspeckle assembly transcript 1 NEAT1 is an essential architectural constituent of paraspeckles in the mammalian nucleus, interacting with Drosophila DBHS RNA-binding proteins such as the splicing factor proline-and glutamine-rich protein SFPQ and the non-POU domain-containing, octamer-binding protein NONO/p54 . . To date, two isoform transcripts of the NEAT1 gene have been identified, namely, the 3.7-kb NEAT1-1 MEN and the 23-kb NEAT1-2 MEN Fig. 1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . .\",\n \"1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . . , promoting cancer formation in mice by dampening oncogene-dependent activation of p53 . . Nevertheless, studies assessing the function of NEAT1 in viral infections are scarce. Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication.\",\n \"Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication. Further investigation showed that NEAT1 promoted RIG-I and DDX60 expression by relocating SFPQ and removing the transcriptional inhibitory effects of SFPQ, which are critical for IFN responses against HTNV infection. We also found that RIG-I signaling, rather than TLR3 and TLR4, accounted for the elevation of HTNV-induced NEAT1. Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection.\",\n \"Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection. As shown in Fig. 1B , the NEAT1 level in the HTNV group was higher than that in the mock group P ϭ 6.86 ϫ 10 Ϫ13 , false discovery rate FDR ϭ 9.75 ϫ 10 Ϫ12 or the 60 Co-inactivated HTNV group P ϭ 1.75 ϫ 10 Ϫ14 , FDR ϭ 3.10 ϫ 10 Ϫ13 ; however, the difference between the 60 Co-inactivated HTNV group and the mock group was not significant P ϭ 0.21034, FDR ϭ 0.58211 . To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig.\",\n \"To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig. 1A , were applied to quantify NEAT1 RNA isoforms by quantitative real-time PCR qRT-PCR . It has been reported that NEAT1-2 rather than NEAT1-1 plays a key regulatory role in paraspeckle formation . , and we also found that elevated NEAT1 levels depend on live HTNV infection rather than 60 Co-inactivated HTNV stimulation Fig. 1C . Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D .\",\n \"Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D . To further investigate whether NEAT1 expression was altered in other cell lines, HEK293, HeLa, and A549 cells were used. All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G .\",\n \"All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G . Similar to the data obtained from HUVECs, NEAT1 was indeed upregulated by HTNV at a multiplicity of infection MOI of 1 beginning at 24 hpi in HUVECs and A549, HEK293, and HeLa cells, and the increasing tendency occurred in a time-dependent manner Fig. 1H . Of note, the NEAT1 elevation at 2 hpi might have been unrelated to the virus but resulted in cellular stress responses. Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I .\",\n \"Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I . NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. The abovedescribed data showed that HTNV infection increased NEAT1, and we wondered how NEAT1 could reciprocally influence HTNV replication. The small interfering RNA siRNA transfection efficiency in HUVECs was confirmed by flow cytometry, and NEAT1 expression was significantly decreased, as assessed by qRT-PCR after RNA interference RNAi Fig. 2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2.\",\n \"2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2. Compared with the cells transfected with control siRNA negative control NC , HUVECs with si-NEAT1 could dramatically promote HTNV NP production, and NP expression seemed to be related to the amount of applied si-NEAT1 Fig. 2B . Intriguingly, depletion of NEAT1-2 alone could mimic the antiviral effects of simultaneous NEAT1-1 and NEAT1-2 silencing Fig. 2C , indicating that NEAT1-2 was critical for the antiviral responses. Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing.\",\n \"Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing. On the other hand, plasmids, of which pCMV-NEAT1-1 is transcribed into the 3.7-kb NEAT1-1 MEN and pCMV-NEAT1-2 is transcribed into the 2-to 3-kb NEAT1-2 MEN , were applied to directly investigate the role of NEAT1 in HTNV infection Fig. 2F . Surprisingly, we found NEAT1-1 overexpression restricted NEAT1-2 transcription Fig. 2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G .\",\n \"2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G . Furthermore, overexpression of NEAT1-2 instead of NEAT1-1 could efficiently suppress HTNV replication Fig. 2H . NEAT1-1 upregulation even aggravated HTNV infection Fig. 2H , which may be the result of downregulation of NEAT1-2. Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig.\",\n \"Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig. 2J were limited in HUVECs in which NEAT1-2 was ectopically expressed in comparison to those transfected with control vector or pCMV-NEAT1-1. These data further showed that NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. Alteration of NEAT1-2 affects HTNV-induced IFN expression in HUVECs. IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . .\",\n \"IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . . It has been reported that the GnT of hantaviruses suppressed IFN- expression of host cells at an early stage of infection . . Here, we also found that HUVECs could not efficiently produce IFN- until 12 hpi at an MOI of 0.1 or until 24 hpi at an MOI of 1 Fig. 3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig.\",\n \"3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig. 3B , suggesting that NEAT1-2 expression increased just before IFN production. We found that expression of endogenous IFN- mRNA was much lower in cells transfected with si-NEAT1-2 at MOIs of both 0.1 Fig. 3C and 1 Fig. 3D than in those transfected with control siRNA NC . In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E .\",\n \"In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E . More importantly, HUVECs transfected with pCMV-NEAT1-2 conspicuously increased IFN- gene expression compared with those cells with vector plasmids at 12 hpi MOI ϭ 1 , demonstrating that NEAT1-2 overexpression accelerated robust IFN responses in host cells against HTNV infection. With a dual luciferase reporter system Twenty-four hours after transfection, the cells expressing FAM were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1.\",\n \"Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the NC group . NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g .\",\n \"NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 0.1 for 48 h. The expression of HTNV NP was measured by Western blotting. C HUVECs were treated as described for panel A, right, but at an MOI of 0.1. In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.\",\n \"In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NC group . D HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group .\",\n \"The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . E HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g .\",\n \"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g . Twenty-four hours after transfection, the cells expressing green fluorescent protein GFP were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with control plasmids vector , pCMV-NEAT1-1, or pCMV-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig.\",\n \"Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig. 3F . These results showed that NEAT1-2 regulated HTNV-induced IFN- expression. To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig.\",\n \"To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig. 3G . In addition, in cells with high NEAT1-2 expression, treatment with neutralizing antibodies NAbs of IFN-␣ and IFN- could counteract the antiviral effects of NEAT1-2 MOI ϭ 1 , and the compensatory effects were dependent on the magnitude of the NAbs. Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production.\",\n \"Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production. PRRs maintain a vital role in the promotion of IFN responses, and we conjectured that NEAT1 might amplify IFN responses by modulating these molecules. TLR3, TLR4, and RIG-I have been shown to recognize HTNV infection . . DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate .\",\n \". DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate . , Here, we found that multiple Toll-like receptors like TLR1, TLR2, TLR3, and TLR4, as well as MDA5, were increased after HTNV infection, but none of them were influenced by silencing NEAT1-2 Fig. 4A . The upregulated RIG-I and DDX60 were blocked in the cells with low NEAT1-2 expression after HTNV infection Fig. 4A . HUVECs with declining NEAT1-2 expression showed gradually decreasing expression of RIG-I and DDX60 Fig. 4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C .\",\n \"4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C . These data indicated that NEAT1-2 could positively modulate RIG-I and DDX60 expression, while the role of RIG-I and DDX60 upon HTNV infection is obscure. We then found that RIG-I and DDX60 colocalized after HTNV infection Fig. 4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown .\",\n \"4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown . Simultaneously knocking down RIG-I and DDX60 significantly promoted HTNV NP expression Fig. 4E , and knockdown of both of them could greatly affect IFN- expression Fig. 4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig.\",\n \"4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig. 4H , indicating that efficient anti-HTNV responses might depend on the interactive effects of DDX60 and RIG-I. More importantly, RIG-I or/and DDX60 overexpression enhanced HTNV-induced IFN- expression, and they had synergistic effects on IFN- production Fig. 4I and J . Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60.\",\n \"Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60. NEAT1 was found to interact with SFPQ by RNA immunoprecipitation RIP after HTNV infection Fig. 5A , indicating that modulatory effects of NEAT1 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively .\",\n \"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1 for 48 h. The expression of HTNV NP was measured by Western blotting. H HUVECs were treated as described for panel F, right. In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.\",\n \"In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . H HUVECs were treated as described for panel F, right. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group .\",\n \"The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . I HUVECs were treated as described for panel F, right. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ.\",\n \"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ. Interestingly, the protein level of SFPQ, as well as another paraspeckle-forming constituent, NONO, remained unchanged after HTNV infection Fig. 5B or after NEAT1 overexpression and knockdown Fig. 5C . However, SFPQ became centralized rather than diffuse in the nucleus after HTNV infection Fig. 5D . The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig.\",\n \"The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig. 5F and G , which might have been related to the increase in RIG-I Fig. 5H and DDX60 Fig. 5I . SFPQ has been suggested to bind to the promoter region of RIG-I and DDX60 ., thus preventing the expression of RIG-I and DDX60. Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription.\",\n \"Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription. Interestingly, overexpression of the S or M segment of HTNV in HEK293 cells failed to induce NEAT1 expression, suggesting that NEAT1 transcription was closely related to live viral replication Fig. 6A . Of note, the upregulation of NEAT1 by HTNV could not be reversed by applying IFN-I neutralizing antibodies Fig. 6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig.\",\n \"6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig. 6C, D, and E or cytokines Fig. 6F and G . We conjectured that NEAT1 expression was related to the activation of PRRs. By knocking down several PRRs, we found that the RIG-I and TLR4 pathways played important roles in HTNV-induced NEAT1 upregulation Fig. 7A . Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C .\",\n \"Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C . Moreover, using STAT1 as a positive control, we found that the transcription factor IRF7, rather than IRF3 and p65, translocated into the nucleus in HTNV-infected HUVECs at 2 dpi Fig. 7D . Furthermore, IRF7 knockdown blocked HTNV-induced NEAT1 upregulation Fig. 7E . Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice.\",\n \"Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice. Although cell-based experiments revealed that NEAT1-2 is a crucial regulator of innate antihantaviral responses, its function in vivo has remained unclear. To address this question, we intravenously injected siRNAs targeting mouse NEAT1-2 at 1 day before HTNV infection. NEAT1-2 expression levels in the liver, kidney, and spleen were reduced at 2 dpi Fig. 8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions.\",\n \"8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions. Body weight loss in NEAT1-2-depleted mice was observed from 2 dpi to 5 dpi, and the IFN production in serum was remarkably decreased in the NEAT1-2 silenced group than those in the NC group at 3 dpi Fig. 8B . As expected, NEAT1-2 knockdown mice showed considerably higher HTNV NP levels in the liver, spleen, and kidney at 3 dpi Fig. 8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D .\",\n \"8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D . In addition, reduced inflammatory cell filtration but increased tissue injury was found in NEAT1-2 knockdown mice during the early stage of infection Fig. 8E . Infiltration of macrophages in the spleen was attenuated Fig. 8F , and the activation of macrophages was also suppressed by flow cytometry; data not shown . Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G .\",\n \"Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G . Nevertheless, NEAT1-2 silencing had no effect on the production of neutralizing antibodies at 7 dpi data not shown . The above-described findings indicated that NEAT1-2 depletion might influence multiple aspects of the innate immune response in HTNV-infected mice. Innate immunity is a phylogenetically ancient and conserved system that counteracts invading microbes, the regulatory mechanism of which is sophisticated and complex. Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . .\",\n \"Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . . In this report, we first demonstrated that NEAT1 was induced by HTNV through the RIG-I-IRF7 pathway and served as positive feedback for RIG-I signaling. Using DGE analysis, we observed upregulated NEAT1 and confirmed its alteration in To further determine the function of NEAT1 after HTNV infection in vivo, mice were injected intravenously with si-NEAT1-2 1 g/g or nontarget control siRNA NC 1 g/g ; 1 day later, they were infected with HTNV 100 LD 50 by intramuscular injection. A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group .\",\n \"A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group . B The effects of NEAT1 on HTNV virulence in mice were determined by body weight loss from 0 to 10 dpi left panel, n ϭ 10 in each group . The IFN- in sera of different groups was measured by ELISA at 3dpi right panel, n ϭ 8 in each group . Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi.\",\n \"Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi. Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . D NEAT1 effects on HTNV infection kinetics at 3 dpi were determined by testing the HTNV titers in livers, spleens, and kidneys. Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group .\",\n \"Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group . After HTNV infection, livers in the NC group showed inflammatory cell infiltration in certain regions, while those in the si-NEAT1-2 group showed slight acute viral hepatitis. Spleens in the NC group showed lymph node hyperplasia, while those in the si-NEAT1-2 group were severely congestive. Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented.\",\n \"Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented. G CD3 ϩ CD8 ϩ IFN-␥ ϩ T cells were analyzed by flow cytometry at 3 dpi, and the results obtained for three mice in each group are presented. different cell lines. To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression.\",\n \"To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression. By virtue of RNAi, the RIG-I-IRF7 pathway was confirmed to be necessary for HTNV-triggered NEAT1 elevation. Recently, large-scale transcriptomic studies identified numerous noncoding transcripts in the mammalian genome, which were speculated to influence diverse biological processes. Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression .\",\n \"Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression . . TLR2 activation or tumor necrosis factor alpha TNF-␣ stimulation induces transcription of the lncRNA THRIL, the downregulation of which impairs TNF-␣ and IL-6 secretion . . TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . .\",\n \". TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . . The lncRNA Lethe, triggered by TNF-␣ and IL-1, acts as a negative feedback regulator of NF-B signaling . . The roles of lncRNAs in host-virus interactions have been progressively unveiled. Various viruses, such as influenza virus IAV , coronavirus, enterovirus, human immunodeficiency virus HIV , hepatitis B virus HBV , hepatitis C virus HCV , Japanese encephalitis virus JEV , and rabies virus, have been reported to activate the transcription of different lncRNAs in host cells 11, . . . .\",\n \". . . Importantly, multiple lncRNAs have been shown to affect the IFN response in recent years and have gradually become hot spots in the field of antiviral research. NeST was shown to enhance IFN-␥ production, controlling the susceptibility of mice to persistent Theiler's virus infection as well as resistance to Salmonella enterica serovar Typhimurium infection . . Both CMPK2 and NRAV were identified as negative regulators of IFN immune reactions. CMPK2, induced by IFN-␣ or HCV infection, suppresses various ISGs, the knockdown of which dramatically blocks HCV replication . . NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . .\",\n \". NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . . Numerous lncRNAs, including lnc-ISG15 and ISR2, respond to IFNs such as ISGs, although their actual function requires further investigation . . Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection.\",\n \"Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection. NEAT1 has been reported to interact with Drosophila DBHS RNA-binding proteins e.g., SFPQ, NONO-p54nrb, and PSPC1 , recruiting them to paraspeckles, a nuclear substructure found in all cultured and primary cells except embryonic stem cells . . The versatile function of NEAT1 is rapidly progressing in multiple areas of biology. NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . .\",\n \"NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . . NEAT1 also participates in neurodegenerative diseases such as Huntington's disease . and seems to potentially contribute to the elevated production of a number of cytokines and chemokines in patients with systemic lupus erythematosus SLE . . Furthermore, poly I·C can activate NEAT1 transcription through the TLR3 pathway, whereas NEAT1 positively regulates IL-8 transcription and potentially affects the expression of multiple ISGs after poly I·C stimulation . . In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . .\",\n \"In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . . However, the role of NEAT1 in hantaviral infection remains unclear. In this report, NEAT1 has been identified as an important regulator of the host innate immune system against HTNV infection. Elevated NEAT1 promotes IFN secretion, most likely by enhancing RIG-I and DDX60 expression. DDX60, a DEXD/H box RNA helicase similar to Saccharomyces cerevisiae Ski2, is induced after viral infection . . DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response.\",\n \". DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response. However, the antiviral effects of DDX60 seem to vary among viruses . . We found that NEAT1-regulated DDX60 was involved in IFN production in response to HTNV infection. In HTNV-infected cells, double-stranded RNA dsRNA could not be detected, and it is unclear how host PRRs, especially RIG-I, recognize HTNV invasion . . Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation.\",\n \". Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation. Of note, we applied multiple cell lines to explore the role of NEAT1 during HTNV infection. HTNV primarily targets vascular endothelial cells in vivo and contributes to the increased vascular permeability and coagulation disorders in HFRS; hence, HUVECs are the most common in vitro cell model to study host innate immunity against HTNV infection or viral pathogenesis . . EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies .\",\n \". EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies . . A549 cells were once used to isolate HTNV, and they were confirmed to be a mature model of infection . . . . . Additionally, Huh 7.0 and Huh 7.5 RIG-I Ϫ cells used in our study have been reported to be infected by HTNV by Lee et al. . and can be used as a cell model to study immune responses against HTNV replication . . Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV.\",\n \". Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV. Using qRT-PCR, Western blotting, and immunofluorescence assays, we have also shown that both HEK293 and HeLa cells can be infected by HTNV. To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production.\",\n \"To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production. Alterations in the relative fluorescence intensity of NP after silencing or overexpressing NEAT1-2 did not seem to be as remarkable as qRT-PCR or Western blot analysis results. The NP spotted and exhibited in the ICW results forms obvious stains that mimic PFU. However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays.\",\n \"However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays. The RNAi studies in vivo are encouraging Fig. 8 , but the NC used by our group was not mutated si-NEAT1-2 i.e., same sense strand, but with a point mutation in the targeting strand . The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling.\",\n \"The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling. After observing that NEAT1 can regulate IFN expression by HTNV infection, we were interested in the function of NEAT1. We noticed that silencing NEAT1-2 or ectopically expressing NEAT1-2 could not inhibit or enhance IFN expression without HTNV infection Fig. 3F , which indicated that NEAT1-2 could not directly affect IFN- expression. This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression.\",\n \"This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression. Recent findings have shown that RIG-I signaling is essential for an efficient polyfunctional T cell response during IAV infection . . Indeed, we found that the function of T cells was suppressed after NEAT1-2 depletion in our animal experiments Fig. 8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection.\",\n \"8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection. HTNV infection induced NEAT1 expression through the RIG-I-IRF7 pathway, while NEAT1 displayed positive feedback for RIG-I signaling. NEAT1 relocated SFPQ from the potential promoter region of several antiviral genes to the paraspeckles, removing the transcriptional inhibitory effects of SFPQ. This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 .\",\n \"This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 . nontarget control i.e., negative control NC or targeted RIG-I and DDX60 were designed by Gene-Pharma as follows: NC, 5=- UUCUUCGAACGUGUCACGUTT-3=; si-RIG-I-1, 5=-GCCCAUUUAAACCAAGAAATT-3=, si-RIG-I-2, 5=-GGUGGAGGAUAUUUGAACUTT-3=, and si-RIG-I-3, 5=-CCCAACGAUAUCAUUUCUTT-3=; si-DDX60-1, 5=-GUCCAGGUGUCAGUUUGAUTT-3=, si-DDX60-2, 5=-CCGAAGUGAAGAAGGUAAATT-3=, and si-DDX60-3, 5=-GAUGGAUGCUAGGAAAUAUTT-3=. The pCMV-NEAT1-1 and pCMV-NEAT1-2 plasmids, which transcribe NEAT1-1 and NEAT1-2, respectively, were provided by Nakagawa Shinichi . . The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . .\",\n \". The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . . Abs against RIG-I, IRF3, IRF7, p65, and STAT1, as well as the neutralizing antibodies against IFN-␣ and IFN-, were purchased from Abcam Cambridge, MA, USA . Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China .\",\n \"Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China . The Abs targeting CD11b, F4/80, CD3, CD8, IFN-␥, inducible nitric oxide synthase iNOS , and CD206 for flow cytometry were purchased from BD Biosciences San Jose, CA, USA . IFN-␣, -, and -␥, TNF-␣, and IL-1 were from PeperoTech Rocky Hill, NJ . ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis.\",\n \"ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis. Total cellular RNAs were extracted with RNAiso TaKaRa, Dalian, China , the concentration of which was measured using a NanoDrop 1000 spectrophotometer. Reverse transcription RT was then performed with PrimeScript RT master mix TaKaRa according to the instructions provided by the manufacturer. Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed.\",\n \"Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed. The qRT-PCR primer sequences for NEAT1, NEAT1-2, IFN-, HTNV S segment, RIG-I, DDX60, -actin, and GAPDH were obtained from previous reports . . The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing.\",\n \". The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing. HUVECs with a confluence of 80% in 6 wells were mock infected or infected with live or 60 Co-inactivated HTNV at an MOI of 1. RNAs were extracted as previously described at 24 hpi, and the quality was analyzed using FastQC software by the Beijing Genomics Institute BGI, Shenzhen, China . Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded.\",\n \"Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded. Tag counts were normalized to TPM transcripts per million by dividing the raw tag count by the total number of tags from each library and multiplying by 1 million. To avoid the possible noise signal from high-throughput sequencing, the genes with average TPM of less than 1 in these three states were excluded. In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01.\",\n \"In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01. Genes were selected as differentially expressed using a P value threshold of 0.01. FISH and immunofluorescence assays IFA . Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature.\",\n \"Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature. Prehybridization was performed with lncRNA FISH probe mix at 37°C for 30 min, and then hybridization was performed by adding NEAT1-2 FISH probe mix and incubating the mixture at 37°C overnight. After washing with 4ϫ, 2ϫ, and 1ϫ SSC, the cell nuclei were stained with DAPI 4=,6-diamidino-2-phenylindole . Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently.\",\n \"Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently. The cells were fixed with 4% PFA for 10 min and permeabilized with 0.1% Triton X-100 for 15 min. Primary Abs were added and incubated at 37°C for 2 h. After five washes with DPBS, secondary Cy3-or fluorescein isothiocyanate FITC -conjugated goat anti-rabbit or goat anti-mouse IgG Sangon, Shanghai, China was added and incubated at 37°C for 2 h. Cell nuclei were stained with DAPI. Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue .\",\n \"Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue . The samples were then boiled at 95°C for 10 min. The lysates were resolved by 10%, 12%, or 15% SDS-PAGE and transferred to polyvinylidene fluoride PVDF membranes Millipore . The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA .\",\n \"The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA . ICW assay. The In-Cell Western ICW assay was performed using an Odyssey imaging system Li-Cor according to the manufacturer's instructions. HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1.\",\n \"HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1. At 48 hpi, HUVECs were washed twice with ice-cold DPBS, fixed with 4% PFA for 10 min, and permeabilized with 1.0% Triton X-100 for 15 min. Cells were added with Li-Cor Odyssey blocking solution at room temperature for 30 min and incubated at 4°C overnight with mouse IgG MAb 1A8 against HTNV NP together with rabbit IgG antibody against -actin, both of which were diluted in PBS containing 3% bovine serum albumin BSA; HyClone . Subsequently, the cells were washed and stained with goat anti-mouse IgG IRDye 800 antibody 1:5,000; Li-Cor and goat anti-rabbit\"\n]"},"document_id":{"kind":"number","value":2652,"string":"2,652"},"id":{"kind":"number","value":4220,"string":"4,220"}}},{"rowIdx":671,"cells":{"question":{"kind":"string","value":"What plays a role in innate immunity to Hantavirus infection?"},"answer":{"kind":"string","value":"long noncoding RNAs (lncRNAs)"},"context_chunks":{"kind":"list like","value":["Hantavirus infection, which causes zoonotic diseases with a high mortality rate in humans, has long been a global public health concern. Over the past decades, accumulating evidence suggests that long noncoding RNAs lncRNAs play key regulatory roles in innate immunity. However, the involvement of host lncRNAs in hantaviral control remains uncharacterized. In this study, we identified the lncRNA NEAT1 as a vital antiviral modulator. NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection.","NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection. Further investigation suggested that NEAT1 served as positive feedback for RIG-I signaling. HTNV infection activated NEAT1 transcription through the RIG-I–IRF7 pathway, whereas NEAT1 removed the transcriptional inhibitory effects of the splicing factor proline- and glutamine-rich protein SFPQ by relocating SFPQ to paraspeckles, thus promoting the expression of RIG-I and DDX60. RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections.","RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections. IMPORTANCE Hantaviruses have attracted worldwide attention as archetypal emerging pathogens. Recently, increasing evidence has highlighted long noncoding RNAs lncRNAs as key regulators of innate immunity; however, their roles in hantavirus infection remain unknown. In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling.","In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling. This lncRNA was induced by HTNV through the RIG-I–IRF7 pathway in a time- and dose-dependent manner and promoted HTNV-induced IFN production by facilitating RIG-I and DDX60 expression. Intriguingly, NEAT1 relocated SFPQ and formed paraspeckles after HTNV infection, which might reverse inhibitive effects of SFPQ on the transcription of RIG-I and DDX60. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections.","To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections. Text: glycoprotein GP , and viral RNA-dependent polymerase protein RdRp , respectively. Humans become infected by inhaling contaminated aerosols or by coming into contact with rodent excreta, and they develop two severe acute diseases, namely, hemorrhagic fever with renal syndrome HFRS and hantavirus pulmonary syndrome HPS . . Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . .",". Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . . Chinese HFRS cases, mainly caused by Hantaan virus HTNV infection, account for approximately 90% of all global cases, with a mortality rate ranging from 0.1 to 15% . . Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions.","Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions. Cellular pathogen recognition receptors PRRs , including Toll-like receptors TLRs and RIG-I like receptors RLRs , can detect distinct pathogen-associated molecular patterns PAMPs and trigger the expression of IFNs and cytokines. RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression.","RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression. lncRNA nuclear paraspeckle assembly transcript 1 NEAT1 is an essential architectural constituent of paraspeckles in the mammalian nucleus, interacting with Drosophila DBHS RNA-binding proteins such as the splicing factor proline-and glutamine-rich protein SFPQ and the non-POU domain-containing, octamer-binding protein NONO/p54 . . To date, two isoform transcripts of the NEAT1 gene have been identified, namely, the 3.7-kb NEAT1-1 MEN and the 23-kb NEAT1-2 MEN Fig. 1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . .","1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . . , promoting cancer formation in mice by dampening oncogene-dependent activation of p53 . . Nevertheless, studies assessing the function of NEAT1 in viral infections are scarce. Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication.","Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication. Further investigation showed that NEAT1 promoted RIG-I and DDX60 expression by relocating SFPQ and removing the transcriptional inhibitory effects of SFPQ, which are critical for IFN responses against HTNV infection. We also found that RIG-I signaling, rather than TLR3 and TLR4, accounted for the elevation of HTNV-induced NEAT1. Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection.","Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection. As shown in Fig. 1B , the NEAT1 level in the HTNV group was higher than that in the mock group P ϭ 6.86 ϫ 10 Ϫ13 , false discovery rate FDR ϭ 9.75 ϫ 10 Ϫ12 or the 60 Co-inactivated HTNV group P ϭ 1.75 ϫ 10 Ϫ14 , FDR ϭ 3.10 ϫ 10 Ϫ13 ; however, the difference between the 60 Co-inactivated HTNV group and the mock group was not significant P ϭ 0.21034, FDR ϭ 0.58211 . To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig.","To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig. 1A , were applied to quantify NEAT1 RNA isoforms by quantitative real-time PCR qRT-PCR . It has been reported that NEAT1-2 rather than NEAT1-1 plays a key regulatory role in paraspeckle formation . , and we also found that elevated NEAT1 levels depend on live HTNV infection rather than 60 Co-inactivated HTNV stimulation Fig. 1C . Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D .","Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D . To further investigate whether NEAT1 expression was altered in other cell lines, HEK293, HeLa, and A549 cells were used. All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G .","All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G . Similar to the data obtained from HUVECs, NEAT1 was indeed upregulated by HTNV at a multiplicity of infection MOI of 1 beginning at 24 hpi in HUVECs and A549, HEK293, and HeLa cells, and the increasing tendency occurred in a time-dependent manner Fig. 1H . Of note, the NEAT1 elevation at 2 hpi might have been unrelated to the virus but resulted in cellular stress responses. Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I .","Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I . NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. The abovedescribed data showed that HTNV infection increased NEAT1, and we wondered how NEAT1 could reciprocally influence HTNV replication. The small interfering RNA siRNA transfection efficiency in HUVECs was confirmed by flow cytometry, and NEAT1 expression was significantly decreased, as assessed by qRT-PCR after RNA interference RNAi Fig. 2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2.","2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2. Compared with the cells transfected with control siRNA negative control NC , HUVECs with si-NEAT1 could dramatically promote HTNV NP production, and NP expression seemed to be related to the amount of applied si-NEAT1 Fig. 2B . Intriguingly, depletion of NEAT1-2 alone could mimic the antiviral effects of simultaneous NEAT1-1 and NEAT1-2 silencing Fig. 2C , indicating that NEAT1-2 was critical for the antiviral responses. Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing.","Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing. On the other hand, plasmids, of which pCMV-NEAT1-1 is transcribed into the 3.7-kb NEAT1-1 MEN and pCMV-NEAT1-2 is transcribed into the 2-to 3-kb NEAT1-2 MEN , were applied to directly investigate the role of NEAT1 in HTNV infection Fig. 2F . Surprisingly, we found NEAT1-1 overexpression restricted NEAT1-2 transcription Fig. 2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G .","2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G . Furthermore, overexpression of NEAT1-2 instead of NEAT1-1 could efficiently suppress HTNV replication Fig. 2H . NEAT1-1 upregulation even aggravated HTNV infection Fig. 2H , which may be the result of downregulation of NEAT1-2. Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig.","Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig. 2J were limited in HUVECs in which NEAT1-2 was ectopically expressed in comparison to those transfected with control vector or pCMV-NEAT1-1. These data further showed that NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. Alteration of NEAT1-2 affects HTNV-induced IFN expression in HUVECs. IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . .","IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . . It has been reported that the GnT of hantaviruses suppressed IFN- expression of host cells at an early stage of infection . . Here, we also found that HUVECs could not efficiently produce IFN- until 12 hpi at an MOI of 0.1 or until 24 hpi at an MOI of 1 Fig. 3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig.","3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig. 3B , suggesting that NEAT1-2 expression increased just before IFN production. We found that expression of endogenous IFN- mRNA was much lower in cells transfected with si-NEAT1-2 at MOIs of both 0.1 Fig. 3C and 1 Fig. 3D than in those transfected with control siRNA NC . In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E .","In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E . More importantly, HUVECs transfected with pCMV-NEAT1-2 conspicuously increased IFN- gene expression compared with those cells with vector plasmids at 12 hpi MOI ϭ 1 , demonstrating that NEAT1-2 overexpression accelerated robust IFN responses in host cells against HTNV infection. With a dual luciferase reporter system Twenty-four hours after transfection, the cells expressing FAM were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1.","Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the NC group . NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g .","NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 0.1 for 48 h. The expression of HTNV NP was measured by Western blotting. C HUVECs were treated as described for panel A, right, but at an MOI of 0.1. In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.","In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NC group . D HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group .","The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . E HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g .","Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g . Twenty-four hours after transfection, the cells expressing green fluorescent protein GFP were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with control plasmids vector , pCMV-NEAT1-1, or pCMV-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig.","Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig. 3F . These results showed that NEAT1-2 regulated HTNV-induced IFN- expression. To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig.","To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig. 3G . In addition, in cells with high NEAT1-2 expression, treatment with neutralizing antibodies NAbs of IFN-␣ and IFN- could counteract the antiviral effects of NEAT1-2 MOI ϭ 1 , and the compensatory effects were dependent on the magnitude of the NAbs. Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production.","Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production. PRRs maintain a vital role in the promotion of IFN responses, and we conjectured that NEAT1 might amplify IFN responses by modulating these molecules. TLR3, TLR4, and RIG-I have been shown to recognize HTNV infection . . DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate .",". DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate . , Here, we found that multiple Toll-like receptors like TLR1, TLR2, TLR3, and TLR4, as well as MDA5, were increased after HTNV infection, but none of them were influenced by silencing NEAT1-2 Fig. 4A . The upregulated RIG-I and DDX60 were blocked in the cells with low NEAT1-2 expression after HTNV infection Fig. 4A . HUVECs with declining NEAT1-2 expression showed gradually decreasing expression of RIG-I and DDX60 Fig. 4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C .","4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C . These data indicated that NEAT1-2 could positively modulate RIG-I and DDX60 expression, while the role of RIG-I and DDX60 upon HTNV infection is obscure. We then found that RIG-I and DDX60 colocalized after HTNV infection Fig. 4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown .","4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown . Simultaneously knocking down RIG-I and DDX60 significantly promoted HTNV NP expression Fig. 4E , and knockdown of both of them could greatly affect IFN- expression Fig. 4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig.","4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig. 4H , indicating that efficient anti-HTNV responses might depend on the interactive effects of DDX60 and RIG-I. More importantly, RIG-I or/and DDX60 overexpression enhanced HTNV-induced IFN- expression, and they had synergistic effects on IFN- production Fig. 4I and J . Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60.","Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60. NEAT1 was found to interact with SFPQ by RNA immunoprecipitation RIP after HTNV infection Fig. 5A , indicating that modulatory effects of NEAT1 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively .","Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1 for 48 h. The expression of HTNV NP was measured by Western blotting. H HUVECs were treated as described for panel F, right. In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.","In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . H HUVECs were treated as described for panel F, right. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group .","The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . I HUVECs were treated as described for panel F, right. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ.","Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ. Interestingly, the protein level of SFPQ, as well as another paraspeckle-forming constituent, NONO, remained unchanged after HTNV infection Fig. 5B or after NEAT1 overexpression and knockdown Fig. 5C . However, SFPQ became centralized rather than diffuse in the nucleus after HTNV infection Fig. 5D . The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig.","The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig. 5F and G , which might have been related to the increase in RIG-I Fig. 5H and DDX60 Fig. 5I . SFPQ has been suggested to bind to the promoter region of RIG-I and DDX60 ., thus preventing the expression of RIG-I and DDX60. Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription.","Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription. Interestingly, overexpression of the S or M segment of HTNV in HEK293 cells failed to induce NEAT1 expression, suggesting that NEAT1 transcription was closely related to live viral replication Fig. 6A . Of note, the upregulation of NEAT1 by HTNV could not be reversed by applying IFN-I neutralizing antibodies Fig. 6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig.","6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig. 6C, D, and E or cytokines Fig. 6F and G . We conjectured that NEAT1 expression was related to the activation of PRRs. By knocking down several PRRs, we found that the RIG-I and TLR4 pathways played important roles in HTNV-induced NEAT1 upregulation Fig. 7A . Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C .","Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C . Moreover, using STAT1 as a positive control, we found that the transcription factor IRF7, rather than IRF3 and p65, translocated into the nucleus in HTNV-infected HUVECs at 2 dpi Fig. 7D . Furthermore, IRF7 knockdown blocked HTNV-induced NEAT1 upregulation Fig. 7E . Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice.","Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice. Although cell-based experiments revealed that NEAT1-2 is a crucial regulator of innate antihantaviral responses, its function in vivo has remained unclear. To address this question, we intravenously injected siRNAs targeting mouse NEAT1-2 at 1 day before HTNV infection. NEAT1-2 expression levels in the liver, kidney, and spleen were reduced at 2 dpi Fig. 8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions.","8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions. Body weight loss in NEAT1-2-depleted mice was observed from 2 dpi to 5 dpi, and the IFN production in serum was remarkably decreased in the NEAT1-2 silenced group than those in the NC group at 3 dpi Fig. 8B . As expected, NEAT1-2 knockdown mice showed considerably higher HTNV NP levels in the liver, spleen, and kidney at 3 dpi Fig. 8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D .","8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D . In addition, reduced inflammatory cell filtration but increased tissue injury was found in NEAT1-2 knockdown mice during the early stage of infection Fig. 8E . Infiltration of macrophages in the spleen was attenuated Fig. 8F , and the activation of macrophages was also suppressed by flow cytometry; data not shown . Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G .","Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G . Nevertheless, NEAT1-2 silencing had no effect on the production of neutralizing antibodies at 7 dpi data not shown . The above-described findings indicated that NEAT1-2 depletion might influence multiple aspects of the innate immune response in HTNV-infected mice. Innate immunity is a phylogenetically ancient and conserved system that counteracts invading microbes, the regulatory mechanism of which is sophisticated and complex. Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . .","Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . . In this report, we first demonstrated that NEAT1 was induced by HTNV through the RIG-I-IRF7 pathway and served as positive feedback for RIG-I signaling. Using DGE analysis, we observed upregulated NEAT1 and confirmed its alteration in To further determine the function of NEAT1 after HTNV infection in vivo, mice were injected intravenously with si-NEAT1-2 1 g/g or nontarget control siRNA NC 1 g/g ; 1 day later, they were infected with HTNV 100 LD 50 by intramuscular injection. A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group .","A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group . B The effects of NEAT1 on HTNV virulence in mice were determined by body weight loss from 0 to 10 dpi left panel, n ϭ 10 in each group . The IFN- in sera of different groups was measured by ELISA at 3dpi right panel, n ϭ 8 in each group . Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi.","Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi. Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . D NEAT1 effects on HTNV infection kinetics at 3 dpi were determined by testing the HTNV titers in livers, spleens, and kidneys. Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group .","Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group . After HTNV infection, livers in the NC group showed inflammatory cell infiltration in certain regions, while those in the si-NEAT1-2 group showed slight acute viral hepatitis. Spleens in the NC group showed lymph node hyperplasia, while those in the si-NEAT1-2 group were severely congestive. Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented.","Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented. G CD3 ϩ CD8 ϩ IFN-␥ ϩ T cells were analyzed by flow cytometry at 3 dpi, and the results obtained for three mice in each group are presented. different cell lines. To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression.","To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression. By virtue of RNAi, the RIG-I-IRF7 pathway was confirmed to be necessary for HTNV-triggered NEAT1 elevation. Recently, large-scale transcriptomic studies identified numerous noncoding transcripts in the mammalian genome, which were speculated to influence diverse biological processes. Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression .","Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression . . TLR2 activation or tumor necrosis factor alpha TNF-␣ stimulation induces transcription of the lncRNA THRIL, the downregulation of which impairs TNF-␣ and IL-6 secretion . . TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . .",". TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . . The lncRNA Lethe, triggered by TNF-␣ and IL-1, acts as a negative feedback regulator of NF-B signaling . . The roles of lncRNAs in host-virus interactions have been progressively unveiled. Various viruses, such as influenza virus IAV , coronavirus, enterovirus, human immunodeficiency virus HIV , hepatitis B virus HBV , hepatitis C virus HCV , Japanese encephalitis virus JEV , and rabies virus, have been reported to activate the transcription of different lncRNAs in host cells 11, . . . .",". . . Importantly, multiple lncRNAs have been shown to affect the IFN response in recent years and have gradually become hot spots in the field of antiviral research. NeST was shown to enhance IFN-␥ production, controlling the susceptibility of mice to persistent Theiler's virus infection as well as resistance to Salmonella enterica serovar Typhimurium infection . . Both CMPK2 and NRAV were identified as negative regulators of IFN immune reactions. CMPK2, induced by IFN-␣ or HCV infection, suppresses various ISGs, the knockdown of which dramatically blocks HCV replication . . NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . .",". NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . . Numerous lncRNAs, including lnc-ISG15 and ISR2, respond to IFNs such as ISGs, although their actual function requires further investigation . . Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection.","Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection. NEAT1 has been reported to interact with Drosophila DBHS RNA-binding proteins e.g., SFPQ, NONO-p54nrb, and PSPC1 , recruiting them to paraspeckles, a nuclear substructure found in all cultured and primary cells except embryonic stem cells . . The versatile function of NEAT1 is rapidly progressing in multiple areas of biology. NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . .","NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . . NEAT1 also participates in neurodegenerative diseases such as Huntington's disease . and seems to potentially contribute to the elevated production of a number of cytokines and chemokines in patients with systemic lupus erythematosus SLE . . Furthermore, poly I·C can activate NEAT1 transcription through the TLR3 pathway, whereas NEAT1 positively regulates IL-8 transcription and potentially affects the expression of multiple ISGs after poly I·C stimulation . . In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . .","In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . . However, the role of NEAT1 in hantaviral infection remains unclear. In this report, NEAT1 has been identified as an important regulator of the host innate immune system against HTNV infection. Elevated NEAT1 promotes IFN secretion, most likely by enhancing RIG-I and DDX60 expression. DDX60, a DEXD/H box RNA helicase similar to Saccharomyces cerevisiae Ski2, is induced after viral infection . . DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response.",". DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response. However, the antiviral effects of DDX60 seem to vary among viruses . . We found that NEAT1-regulated DDX60 was involved in IFN production in response to HTNV infection. In HTNV-infected cells, double-stranded RNA dsRNA could not be detected, and it is unclear how host PRRs, especially RIG-I, recognize HTNV invasion . . Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation.",". Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation. Of note, we applied multiple cell lines to explore the role of NEAT1 during HTNV infection. HTNV primarily targets vascular endothelial cells in vivo and contributes to the increased vascular permeability and coagulation disorders in HFRS; hence, HUVECs are the most common in vitro cell model to study host innate immunity against HTNV infection or viral pathogenesis . . EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies .",". EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies . . A549 cells were once used to isolate HTNV, and they were confirmed to be a mature model of infection . . . . . Additionally, Huh 7.0 and Huh 7.5 RIG-I Ϫ cells used in our study have been reported to be infected by HTNV by Lee et al. . and can be used as a cell model to study immune responses against HTNV replication . . Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV.",". Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV. Using qRT-PCR, Western blotting, and immunofluorescence assays, we have also shown that both HEK293 and HeLa cells can be infected by HTNV. To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production.","To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production. Alterations in the relative fluorescence intensity of NP after silencing or overexpressing NEAT1-2 did not seem to be as remarkable as qRT-PCR or Western blot analysis results. The NP spotted and exhibited in the ICW results forms obvious stains that mimic PFU. However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays.","However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays. The RNAi studies in vivo are encouraging Fig. 8 , but the NC used by our group was not mutated si-NEAT1-2 i.e., same sense strand, but with a point mutation in the targeting strand . The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling.","The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling. After observing that NEAT1 can regulate IFN expression by HTNV infection, we were interested in the function of NEAT1. We noticed that silencing NEAT1-2 or ectopically expressing NEAT1-2 could not inhibit or enhance IFN expression without HTNV infection Fig. 3F , which indicated that NEAT1-2 could not directly affect IFN- expression. This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression.","This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression. Recent findings have shown that RIG-I signaling is essential for an efficient polyfunctional T cell response during IAV infection . . Indeed, we found that the function of T cells was suppressed after NEAT1-2 depletion in our animal experiments Fig. 8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection.","8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection. HTNV infection induced NEAT1 expression through the RIG-I-IRF7 pathway, while NEAT1 displayed positive feedback for RIG-I signaling. NEAT1 relocated SFPQ from the potential promoter region of several antiviral genes to the paraspeckles, removing the transcriptional inhibitory effects of SFPQ. This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 .","This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 . nontarget control i.e., negative control NC or targeted RIG-I and DDX60 were designed by Gene-Pharma as follows: NC, 5=- UUCUUCGAACGUGUCACGUTT-3=; si-RIG-I-1, 5=-GCCCAUUUAAACCAAGAAATT-3=, si-RIG-I-2, 5=-GGUGGAGGAUAUUUGAACUTT-3=, and si-RIG-I-3, 5=-CCCAACGAUAUCAUUUCUTT-3=; si-DDX60-1, 5=-GUCCAGGUGUCAGUUUGAUTT-3=, si-DDX60-2, 5=-CCGAAGUGAAGAAGGUAAATT-3=, and si-DDX60-3, 5=-GAUGGAUGCUAGGAAAUAUTT-3=. The pCMV-NEAT1-1 and pCMV-NEAT1-2 plasmids, which transcribe NEAT1-1 and NEAT1-2, respectively, were provided by Nakagawa Shinichi . . The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . .",". The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . . Abs against RIG-I, IRF3, IRF7, p65, and STAT1, as well as the neutralizing antibodies against IFN-␣ and IFN-, were purchased from Abcam Cambridge, MA, USA . Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China .","Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China . The Abs targeting CD11b, F4/80, CD3, CD8, IFN-␥, inducible nitric oxide synthase iNOS , and CD206 for flow cytometry were purchased from BD Biosciences San Jose, CA, USA . IFN-␣, -, and -␥, TNF-␣, and IL-1 were from PeperoTech Rocky Hill, NJ . ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis.","ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis. Total cellular RNAs were extracted with RNAiso TaKaRa, Dalian, China , the concentration of which was measured using a NanoDrop 1000 spectrophotometer. Reverse transcription RT was then performed with PrimeScript RT master mix TaKaRa according to the instructions provided by the manufacturer. Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed.","Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed. The qRT-PCR primer sequences for NEAT1, NEAT1-2, IFN-, HTNV S segment, RIG-I, DDX60, -actin, and GAPDH were obtained from previous reports . . The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing.",". The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing. HUVECs with a confluence of 80% in 6 wells were mock infected or infected with live or 60 Co-inactivated HTNV at an MOI of 1. RNAs were extracted as previously described at 24 hpi, and the quality was analyzed using FastQC software by the Beijing Genomics Institute BGI, Shenzhen, China . Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded.","Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded. Tag counts were normalized to TPM transcripts per million by dividing the raw tag count by the total number of tags from each library and multiplying by 1 million. To avoid the possible noise signal from high-throughput sequencing, the genes with average TPM of less than 1 in these three states were excluded. In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01.","In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01. Genes were selected as differentially expressed using a P value threshold of 0.01. FISH and immunofluorescence assays IFA . Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature.","Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature. Prehybridization was performed with lncRNA FISH probe mix at 37°C for 30 min, and then hybridization was performed by adding NEAT1-2 FISH probe mix and incubating the mixture at 37°C overnight. After washing with 4ϫ, 2ϫ, and 1ϫ SSC, the cell nuclei were stained with DAPI 4=,6-diamidino-2-phenylindole . Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently.","Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently. The cells were fixed with 4% PFA for 10 min and permeabilized with 0.1% Triton X-100 for 15 min. Primary Abs were added and incubated at 37°C for 2 h. After five washes with DPBS, secondary Cy3-or fluorescein isothiocyanate FITC -conjugated goat anti-rabbit or goat anti-mouse IgG Sangon, Shanghai, China was added and incubated at 37°C for 2 h. Cell nuclei were stained with DAPI. Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue .","Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue . The samples were then boiled at 95°C for 10 min. The lysates were resolved by 10%, 12%, or 15% SDS-PAGE and transferred to polyvinylidene fluoride PVDF membranes Millipore . The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA .","The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA . ICW assay. The In-Cell Western ICW assay was performed using an Odyssey imaging system Li-Cor according to the manufacturer's instructions. HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1.","HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1. At 48 hpi, HUVECs were washed twice with ice-cold DPBS, fixed with 4% PFA for 10 min, and permeabilized with 1.0% Triton X-100 for 15 min. Cells were added with Li-Cor Odyssey blocking solution at room temperature for 30 min and incubated at 4°C overnight with mouse IgG MAb 1A8 against HTNV NP together with rabbit IgG antibody against -actin, both of which were diluted in PBS containing 3% bovine serum albumin BSA; HyClone . Subsequently, the cells were washed and stained with goat anti-mouse IgG IRDye 800 antibody 1:5,000; Li-Cor and goat anti-rabbit"],"string":"[\n \"Hantavirus infection, which causes zoonotic diseases with a high mortality rate in humans, has long been a global public health concern. Over the past decades, accumulating evidence suggests that long noncoding RNAs lncRNAs play key regulatory roles in innate immunity. However, the involvement of host lncRNAs in hantaviral control remains uncharacterized. In this study, we identified the lncRNA NEAT1 as a vital antiviral modulator. NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection.\",\n \"NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection. Further investigation suggested that NEAT1 served as positive feedback for RIG-I signaling. HTNV infection activated NEAT1 transcription through the RIG-I–IRF7 pathway, whereas NEAT1 removed the transcriptional inhibitory effects of the splicing factor proline- and glutamine-rich protein SFPQ by relocating SFPQ to paraspeckles, thus promoting the expression of RIG-I and DDX60. RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections.\",\n \"RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections. IMPORTANCE Hantaviruses have attracted worldwide attention as archetypal emerging pathogens. Recently, increasing evidence has highlighted long noncoding RNAs lncRNAs as key regulators of innate immunity; however, their roles in hantavirus infection remain unknown. In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling.\",\n \"In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling. This lncRNA was induced by HTNV through the RIG-I–IRF7 pathway in a time- and dose-dependent manner and promoted HTNV-induced IFN production by facilitating RIG-I and DDX60 expression. Intriguingly, NEAT1 relocated SFPQ and formed paraspeckles after HTNV infection, which might reverse inhibitive effects of SFPQ on the transcription of RIG-I and DDX60. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections.\",\n \"To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections. Text: glycoprotein GP , and viral RNA-dependent polymerase protein RdRp , respectively. Humans become infected by inhaling contaminated aerosols or by coming into contact with rodent excreta, and they develop two severe acute diseases, namely, hemorrhagic fever with renal syndrome HFRS and hantavirus pulmonary syndrome HPS . . Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . .\",\n \". Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . . Chinese HFRS cases, mainly caused by Hantaan virus HTNV infection, account for approximately 90% of all global cases, with a mortality rate ranging from 0.1 to 15% . . Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions.\",\n \"Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions. Cellular pathogen recognition receptors PRRs , including Toll-like receptors TLRs and RIG-I like receptors RLRs , can detect distinct pathogen-associated molecular patterns PAMPs and trigger the expression of IFNs and cytokines. RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression.\",\n \"RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression. lncRNA nuclear paraspeckle assembly transcript 1 NEAT1 is an essential architectural constituent of paraspeckles in the mammalian nucleus, interacting with Drosophila DBHS RNA-binding proteins such as the splicing factor proline-and glutamine-rich protein SFPQ and the non-POU domain-containing, octamer-binding protein NONO/p54 . . To date, two isoform transcripts of the NEAT1 gene have been identified, namely, the 3.7-kb NEAT1-1 MEN and the 23-kb NEAT1-2 MEN Fig. 1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . .\",\n \"1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . . , promoting cancer formation in mice by dampening oncogene-dependent activation of p53 . . Nevertheless, studies assessing the function of NEAT1 in viral infections are scarce. Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication.\",\n \"Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication. Further investigation showed that NEAT1 promoted RIG-I and DDX60 expression by relocating SFPQ and removing the transcriptional inhibitory effects of SFPQ, which are critical for IFN responses against HTNV infection. We also found that RIG-I signaling, rather than TLR3 and TLR4, accounted for the elevation of HTNV-induced NEAT1. Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection.\",\n \"Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection. As shown in Fig. 1B , the NEAT1 level in the HTNV group was higher than that in the mock group P ϭ 6.86 ϫ 10 Ϫ13 , false discovery rate FDR ϭ 9.75 ϫ 10 Ϫ12 or the 60 Co-inactivated HTNV group P ϭ 1.75 ϫ 10 Ϫ14 , FDR ϭ 3.10 ϫ 10 Ϫ13 ; however, the difference between the 60 Co-inactivated HTNV group and the mock group was not significant P ϭ 0.21034, FDR ϭ 0.58211 . To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig.\",\n \"To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig. 1A , were applied to quantify NEAT1 RNA isoforms by quantitative real-time PCR qRT-PCR . It has been reported that NEAT1-2 rather than NEAT1-1 plays a key regulatory role in paraspeckle formation . , and we also found that elevated NEAT1 levels depend on live HTNV infection rather than 60 Co-inactivated HTNV stimulation Fig. 1C . Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D .\",\n \"Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D . To further investigate whether NEAT1 expression was altered in other cell lines, HEK293, HeLa, and A549 cells were used. All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G .\",\n \"All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G . Similar to the data obtained from HUVECs, NEAT1 was indeed upregulated by HTNV at a multiplicity of infection MOI of 1 beginning at 24 hpi in HUVECs and A549, HEK293, and HeLa cells, and the increasing tendency occurred in a time-dependent manner Fig. 1H . Of note, the NEAT1 elevation at 2 hpi might have been unrelated to the virus but resulted in cellular stress responses. Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I .\",\n \"Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I . NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. The abovedescribed data showed that HTNV infection increased NEAT1, and we wondered how NEAT1 could reciprocally influence HTNV replication. The small interfering RNA siRNA transfection efficiency in HUVECs was confirmed by flow cytometry, and NEAT1 expression was significantly decreased, as assessed by qRT-PCR after RNA interference RNAi Fig. 2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2.\",\n \"2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2. Compared with the cells transfected with control siRNA negative control NC , HUVECs with si-NEAT1 could dramatically promote HTNV NP production, and NP expression seemed to be related to the amount of applied si-NEAT1 Fig. 2B . Intriguingly, depletion of NEAT1-2 alone could mimic the antiviral effects of simultaneous NEAT1-1 and NEAT1-2 silencing Fig. 2C , indicating that NEAT1-2 was critical for the antiviral responses. Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing.\",\n \"Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing. On the other hand, plasmids, of which pCMV-NEAT1-1 is transcribed into the 3.7-kb NEAT1-1 MEN and pCMV-NEAT1-2 is transcribed into the 2-to 3-kb NEAT1-2 MEN , were applied to directly investigate the role of NEAT1 in HTNV infection Fig. 2F . Surprisingly, we found NEAT1-1 overexpression restricted NEAT1-2 transcription Fig. 2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G .\",\n \"2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G . Furthermore, overexpression of NEAT1-2 instead of NEAT1-1 could efficiently suppress HTNV replication Fig. 2H . NEAT1-1 upregulation even aggravated HTNV infection Fig. 2H , which may be the result of downregulation of NEAT1-2. Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig.\",\n \"Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig. 2J were limited in HUVECs in which NEAT1-2 was ectopically expressed in comparison to those transfected with control vector or pCMV-NEAT1-1. These data further showed that NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. Alteration of NEAT1-2 affects HTNV-induced IFN expression in HUVECs. IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . .\",\n \"IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . . It has been reported that the GnT of hantaviruses suppressed IFN- expression of host cells at an early stage of infection . . Here, we also found that HUVECs could not efficiently produce IFN- until 12 hpi at an MOI of 0.1 or until 24 hpi at an MOI of 1 Fig. 3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig.\",\n \"3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig. 3B , suggesting that NEAT1-2 expression increased just before IFN production. We found that expression of endogenous IFN- mRNA was much lower in cells transfected with si-NEAT1-2 at MOIs of both 0.1 Fig. 3C and 1 Fig. 3D than in those transfected with control siRNA NC . In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E .\",\n \"In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E . More importantly, HUVECs transfected with pCMV-NEAT1-2 conspicuously increased IFN- gene expression compared with those cells with vector plasmids at 12 hpi MOI ϭ 1 , demonstrating that NEAT1-2 overexpression accelerated robust IFN responses in host cells against HTNV infection. With a dual luciferase reporter system Twenty-four hours after transfection, the cells expressing FAM were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1.\",\n \"Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the NC group . NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g .\",\n \"NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 0.1 for 48 h. The expression of HTNV NP was measured by Western blotting. C HUVECs were treated as described for panel A, right, but at an MOI of 0.1. In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.\",\n \"In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NC group . D HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group .\",\n \"The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . E HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g .\",\n \"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g . Twenty-four hours after transfection, the cells expressing green fluorescent protein GFP were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with control plasmids vector , pCMV-NEAT1-1, or pCMV-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig.\",\n \"Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig. 3F . These results showed that NEAT1-2 regulated HTNV-induced IFN- expression. To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig.\",\n \"To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig. 3G . In addition, in cells with high NEAT1-2 expression, treatment with neutralizing antibodies NAbs of IFN-␣ and IFN- could counteract the antiviral effects of NEAT1-2 MOI ϭ 1 , and the compensatory effects were dependent on the magnitude of the NAbs. Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production.\",\n \"Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production. PRRs maintain a vital role in the promotion of IFN responses, and we conjectured that NEAT1 might amplify IFN responses by modulating these molecules. TLR3, TLR4, and RIG-I have been shown to recognize HTNV infection . . DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate .\",\n \". DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate . , Here, we found that multiple Toll-like receptors like TLR1, TLR2, TLR3, and TLR4, as well as MDA5, were increased after HTNV infection, but none of them were influenced by silencing NEAT1-2 Fig. 4A . The upregulated RIG-I and DDX60 were blocked in the cells with low NEAT1-2 expression after HTNV infection Fig. 4A . HUVECs with declining NEAT1-2 expression showed gradually decreasing expression of RIG-I and DDX60 Fig. 4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C .\",\n \"4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C . These data indicated that NEAT1-2 could positively modulate RIG-I and DDX60 expression, while the role of RIG-I and DDX60 upon HTNV infection is obscure. We then found that RIG-I and DDX60 colocalized after HTNV infection Fig. 4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown .\",\n \"4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown . Simultaneously knocking down RIG-I and DDX60 significantly promoted HTNV NP expression Fig. 4E , and knockdown of both of them could greatly affect IFN- expression Fig. 4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig.\",\n \"4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig. 4H , indicating that efficient anti-HTNV responses might depend on the interactive effects of DDX60 and RIG-I. More importantly, RIG-I or/and DDX60 overexpression enhanced HTNV-induced IFN- expression, and they had synergistic effects on IFN- production Fig. 4I and J . Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60.\",\n \"Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60. NEAT1 was found to interact with SFPQ by RNA immunoprecipitation RIP after HTNV infection Fig. 5A , indicating that modulatory effects of NEAT1 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively .\",\n \"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1 for 48 h. The expression of HTNV NP was measured by Western blotting. H HUVECs were treated as described for panel F, right. In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.\",\n \"In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . H HUVECs were treated as described for panel F, right. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group .\",\n \"The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . I HUVECs were treated as described for panel F, right. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ.\",\n \"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ. Interestingly, the protein level of SFPQ, as well as another paraspeckle-forming constituent, NONO, remained unchanged after HTNV infection Fig. 5B or after NEAT1 overexpression and knockdown Fig. 5C . However, SFPQ became centralized rather than diffuse in the nucleus after HTNV infection Fig. 5D . The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig.\",\n \"The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig. 5F and G , which might have been related to the increase in RIG-I Fig. 5H and DDX60 Fig. 5I . SFPQ has been suggested to bind to the promoter region of RIG-I and DDX60 ., thus preventing the expression of RIG-I and DDX60. Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription.\",\n \"Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription. Interestingly, overexpression of the S or M segment of HTNV in HEK293 cells failed to induce NEAT1 expression, suggesting that NEAT1 transcription was closely related to live viral replication Fig. 6A . Of note, the upregulation of NEAT1 by HTNV could not be reversed by applying IFN-I neutralizing antibodies Fig. 6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig.\",\n \"6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig. 6C, D, and E or cytokines Fig. 6F and G . We conjectured that NEAT1 expression was related to the activation of PRRs. By knocking down several PRRs, we found that the RIG-I and TLR4 pathways played important roles in HTNV-induced NEAT1 upregulation Fig. 7A . Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C .\",\n \"Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C . Moreover, using STAT1 as a positive control, we found that the transcription factor IRF7, rather than IRF3 and p65, translocated into the nucleus in HTNV-infected HUVECs at 2 dpi Fig. 7D . Furthermore, IRF7 knockdown blocked HTNV-induced NEAT1 upregulation Fig. 7E . Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice.\",\n \"Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice. Although cell-based experiments revealed that NEAT1-2 is a crucial regulator of innate antihantaviral responses, its function in vivo has remained unclear. To address this question, we intravenously injected siRNAs targeting mouse NEAT1-2 at 1 day before HTNV infection. NEAT1-2 expression levels in the liver, kidney, and spleen were reduced at 2 dpi Fig. 8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions.\",\n \"8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions. Body weight loss in NEAT1-2-depleted mice was observed from 2 dpi to 5 dpi, and the IFN production in serum was remarkably decreased in the NEAT1-2 silenced group than those in the NC group at 3 dpi Fig. 8B . As expected, NEAT1-2 knockdown mice showed considerably higher HTNV NP levels in the liver, spleen, and kidney at 3 dpi Fig. 8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D .\",\n \"8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D . In addition, reduced inflammatory cell filtration but increased tissue injury was found in NEAT1-2 knockdown mice during the early stage of infection Fig. 8E . Infiltration of macrophages in the spleen was attenuated Fig. 8F , and the activation of macrophages was also suppressed by flow cytometry; data not shown . Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G .\",\n \"Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G . Nevertheless, NEAT1-2 silencing had no effect on the production of neutralizing antibodies at 7 dpi data not shown . The above-described findings indicated that NEAT1-2 depletion might influence multiple aspects of the innate immune response in HTNV-infected mice. Innate immunity is a phylogenetically ancient and conserved system that counteracts invading microbes, the regulatory mechanism of which is sophisticated and complex. Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . .\",\n \"Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . . In this report, we first demonstrated that NEAT1 was induced by HTNV through the RIG-I-IRF7 pathway and served as positive feedback for RIG-I signaling. Using DGE analysis, we observed upregulated NEAT1 and confirmed its alteration in To further determine the function of NEAT1 after HTNV infection in vivo, mice were injected intravenously with si-NEAT1-2 1 g/g or nontarget control siRNA NC 1 g/g ; 1 day later, they were infected with HTNV 100 LD 50 by intramuscular injection. A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group .\",\n \"A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group . B The effects of NEAT1 on HTNV virulence in mice were determined by body weight loss from 0 to 10 dpi left panel, n ϭ 10 in each group . The IFN- in sera of different groups was measured by ELISA at 3dpi right panel, n ϭ 8 in each group . Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi.\",\n \"Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi. Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . D NEAT1 effects on HTNV infection kinetics at 3 dpi were determined by testing the HTNV titers in livers, spleens, and kidneys. Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group .\",\n \"Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group . After HTNV infection, livers in the NC group showed inflammatory cell infiltration in certain regions, while those in the si-NEAT1-2 group showed slight acute viral hepatitis. Spleens in the NC group showed lymph node hyperplasia, while those in the si-NEAT1-2 group were severely congestive. Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented.\",\n \"Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented. G CD3 ϩ CD8 ϩ IFN-␥ ϩ T cells were analyzed by flow cytometry at 3 dpi, and the results obtained for three mice in each group are presented. different cell lines. To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression.\",\n \"To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression. By virtue of RNAi, the RIG-I-IRF7 pathway was confirmed to be necessary for HTNV-triggered NEAT1 elevation. Recently, large-scale transcriptomic studies identified numerous noncoding transcripts in the mammalian genome, which were speculated to influence diverse biological processes. Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression .\",\n \"Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression . . TLR2 activation or tumor necrosis factor alpha TNF-␣ stimulation induces transcription of the lncRNA THRIL, the downregulation of which impairs TNF-␣ and IL-6 secretion . . TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . .\",\n \". TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . . The lncRNA Lethe, triggered by TNF-␣ and IL-1, acts as a negative feedback regulator of NF-B signaling . . The roles of lncRNAs in host-virus interactions have been progressively unveiled. Various viruses, such as influenza virus IAV , coronavirus, enterovirus, human immunodeficiency virus HIV , hepatitis B virus HBV , hepatitis C virus HCV , Japanese encephalitis virus JEV , and rabies virus, have been reported to activate the transcription of different lncRNAs in host cells 11, . . . .\",\n \". . . Importantly, multiple lncRNAs have been shown to affect the IFN response in recent years and have gradually become hot spots in the field of antiviral research. NeST was shown to enhance IFN-␥ production, controlling the susceptibility of mice to persistent Theiler's virus infection as well as resistance to Salmonella enterica serovar Typhimurium infection . . Both CMPK2 and NRAV were identified as negative regulators of IFN immune reactions. CMPK2, induced by IFN-␣ or HCV infection, suppresses various ISGs, the knockdown of which dramatically blocks HCV replication . . NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . .\",\n \". NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . . Numerous lncRNAs, including lnc-ISG15 and ISR2, respond to IFNs such as ISGs, although their actual function requires further investigation . . Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection.\",\n \"Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection. NEAT1 has been reported to interact with Drosophila DBHS RNA-binding proteins e.g., SFPQ, NONO-p54nrb, and PSPC1 , recruiting them to paraspeckles, a nuclear substructure found in all cultured and primary cells except embryonic stem cells . . The versatile function of NEAT1 is rapidly progressing in multiple areas of biology. NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . .\",\n \"NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . . NEAT1 also participates in neurodegenerative diseases such as Huntington's disease . and seems to potentially contribute to the elevated production of a number of cytokines and chemokines in patients with systemic lupus erythematosus SLE . . Furthermore, poly I·C can activate NEAT1 transcription through the TLR3 pathway, whereas NEAT1 positively regulates IL-8 transcription and potentially affects the expression of multiple ISGs after poly I·C stimulation . . In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . .\",\n \"In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . . However, the role of NEAT1 in hantaviral infection remains unclear. In this report, NEAT1 has been identified as an important regulator of the host innate immune system against HTNV infection. Elevated NEAT1 promotes IFN secretion, most likely by enhancing RIG-I and DDX60 expression. DDX60, a DEXD/H box RNA helicase similar to Saccharomyces cerevisiae Ski2, is induced after viral infection . . DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response.\",\n \". DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response. However, the antiviral effects of DDX60 seem to vary among viruses . . We found that NEAT1-regulated DDX60 was involved in IFN production in response to HTNV infection. In HTNV-infected cells, double-stranded RNA dsRNA could not be detected, and it is unclear how host PRRs, especially RIG-I, recognize HTNV invasion . . Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation.\",\n \". Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation. Of note, we applied multiple cell lines to explore the role of NEAT1 during HTNV infection. HTNV primarily targets vascular endothelial cells in vivo and contributes to the increased vascular permeability and coagulation disorders in HFRS; hence, HUVECs are the most common in vitro cell model to study host innate immunity against HTNV infection or viral pathogenesis . . EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies .\",\n \". EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies . . A549 cells were once used to isolate HTNV, and they were confirmed to be a mature model of infection . . . . . Additionally, Huh 7.0 and Huh 7.5 RIG-I Ϫ cells used in our study have been reported to be infected by HTNV by Lee et al. . and can be used as a cell model to study immune responses against HTNV replication . . Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV.\",\n \". Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV. Using qRT-PCR, Western blotting, and immunofluorescence assays, we have also shown that both HEK293 and HeLa cells can be infected by HTNV. To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production.\",\n \"To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production. Alterations in the relative fluorescence intensity of NP after silencing or overexpressing NEAT1-2 did not seem to be as remarkable as qRT-PCR or Western blot analysis results. The NP spotted and exhibited in the ICW results forms obvious stains that mimic PFU. However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays.\",\n \"However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays. The RNAi studies in vivo are encouraging Fig. 8 , but the NC used by our group was not mutated si-NEAT1-2 i.e., same sense strand, but with a point mutation in the targeting strand . The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling.\",\n \"The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling. After observing that NEAT1 can regulate IFN expression by HTNV infection, we were interested in the function of NEAT1. We noticed that silencing NEAT1-2 or ectopically expressing NEAT1-2 could not inhibit or enhance IFN expression without HTNV infection Fig. 3F , which indicated that NEAT1-2 could not directly affect IFN- expression. This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression.\",\n \"This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression. Recent findings have shown that RIG-I signaling is essential for an efficient polyfunctional T cell response during IAV infection . . Indeed, we found that the function of T cells was suppressed after NEAT1-2 depletion in our animal experiments Fig. 8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection.\",\n \"8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection. HTNV infection induced NEAT1 expression through the RIG-I-IRF7 pathway, while NEAT1 displayed positive feedback for RIG-I signaling. NEAT1 relocated SFPQ from the potential promoter region of several antiviral genes to the paraspeckles, removing the transcriptional inhibitory effects of SFPQ. This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 .\",\n \"This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 . nontarget control i.e., negative control NC or targeted RIG-I and DDX60 were designed by Gene-Pharma as follows: NC, 5=- UUCUUCGAACGUGUCACGUTT-3=; si-RIG-I-1, 5=-GCCCAUUUAAACCAAGAAATT-3=, si-RIG-I-2, 5=-GGUGGAGGAUAUUUGAACUTT-3=, and si-RIG-I-3, 5=-CCCAACGAUAUCAUUUCUTT-3=; si-DDX60-1, 5=-GUCCAGGUGUCAGUUUGAUTT-3=, si-DDX60-2, 5=-CCGAAGUGAAGAAGGUAAATT-3=, and si-DDX60-3, 5=-GAUGGAUGCUAGGAAAUAUTT-3=. The pCMV-NEAT1-1 and pCMV-NEAT1-2 plasmids, which transcribe NEAT1-1 and NEAT1-2, respectively, were provided by Nakagawa Shinichi . . The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . .\",\n \". The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . . Abs against RIG-I, IRF3, IRF7, p65, and STAT1, as well as the neutralizing antibodies against IFN-␣ and IFN-, were purchased from Abcam Cambridge, MA, USA . Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China .\",\n \"Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China . The Abs targeting CD11b, F4/80, CD3, CD8, IFN-␥, inducible nitric oxide synthase iNOS , and CD206 for flow cytometry were purchased from BD Biosciences San Jose, CA, USA . IFN-␣, -, and -␥, TNF-␣, and IL-1 were from PeperoTech Rocky Hill, NJ . ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis.\",\n \"ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis. Total cellular RNAs were extracted with RNAiso TaKaRa, Dalian, China , the concentration of which was measured using a NanoDrop 1000 spectrophotometer. Reverse transcription RT was then performed with PrimeScript RT master mix TaKaRa according to the instructions provided by the manufacturer. Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed.\",\n \"Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed. The qRT-PCR primer sequences for NEAT1, NEAT1-2, IFN-, HTNV S segment, RIG-I, DDX60, -actin, and GAPDH were obtained from previous reports . . The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing.\",\n \". The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing. HUVECs with a confluence of 80% in 6 wells were mock infected or infected with live or 60 Co-inactivated HTNV at an MOI of 1. RNAs were extracted as previously described at 24 hpi, and the quality was analyzed using FastQC software by the Beijing Genomics Institute BGI, Shenzhen, China . Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded.\",\n \"Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded. Tag counts were normalized to TPM transcripts per million by dividing the raw tag count by the total number of tags from each library and multiplying by 1 million. To avoid the possible noise signal from high-throughput sequencing, the genes with average TPM of less than 1 in these three states were excluded. In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01.\",\n \"In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01. Genes were selected as differentially expressed using a P value threshold of 0.01. FISH and immunofluorescence assays IFA . Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature.\",\n \"Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature. Prehybridization was performed with lncRNA FISH probe mix at 37°C for 30 min, and then hybridization was performed by adding NEAT1-2 FISH probe mix and incubating the mixture at 37°C overnight. After washing with 4ϫ, 2ϫ, and 1ϫ SSC, the cell nuclei were stained with DAPI 4=,6-diamidino-2-phenylindole . Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently.\",\n \"Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently. The cells were fixed with 4% PFA for 10 min and permeabilized with 0.1% Triton X-100 for 15 min. Primary Abs were added and incubated at 37°C for 2 h. After five washes with DPBS, secondary Cy3-or fluorescein isothiocyanate FITC -conjugated goat anti-rabbit or goat anti-mouse IgG Sangon, Shanghai, China was added and incubated at 37°C for 2 h. Cell nuclei were stained with DAPI. Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue .\",\n \"Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue . The samples were then boiled at 95°C for 10 min. The lysates were resolved by 10%, 12%, or 15% SDS-PAGE and transferred to polyvinylidene fluoride PVDF membranes Millipore . The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA .\",\n \"The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA . ICW assay. The In-Cell Western ICW assay was performed using an Odyssey imaging system Li-Cor according to the manufacturer's instructions. HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1.\",\n \"HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1. At 48 hpi, HUVECs were washed twice with ice-cold DPBS, fixed with 4% PFA for 10 min, and permeabilized with 1.0% Triton X-100 for 15 min. Cells were added with Li-Cor Odyssey blocking solution at room temperature for 30 min and incubated at 4°C overnight with mouse IgG MAb 1A8 against HTNV NP together with rabbit IgG antibody against -actin, both of which were diluted in PBS containing 3% bovine serum albumin BSA; HyClone . Subsequently, the cells were washed and stained with goat anti-mouse IgG IRDye 800 antibody 1:5,000; Li-Cor and goat anti-rabbit\"\n]"},"document_id":{"kind":"number","value":2652,"string":"2,652"},"id":{"kind":"number","value":5311,"string":"5,311"}}},{"rowIdx":672,"cells":{"question":{"kind":"string","value":"What evidence suggests that RIG-I and DDX60 collaborate to exert antiviral effects?"},"answer":{"kind":"string","value":"colocalized after HTNV infection"},"context_chunks":{"kind":"list like","value":["Hantavirus infection, which causes zoonotic diseases with a high mortality rate in humans, has long been a global public health concern. Over the past decades, accumulating evidence suggests that long noncoding RNAs lncRNAs play key regulatory roles in innate immunity. However, the involvement of host lncRNAs in hantaviral control remains uncharacterized. In this study, we identified the lncRNA NEAT1 as a vital antiviral modulator. NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection.","NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection. Further investigation suggested that NEAT1 served as positive feedback for RIG-I signaling. HTNV infection activated NEAT1 transcription through the RIG-I–IRF7 pathway, whereas NEAT1 removed the transcriptional inhibitory effects of the splicing factor proline- and glutamine-rich protein SFPQ by relocating SFPQ to paraspeckles, thus promoting the expression of RIG-I and DDX60. RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections.","RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections. IMPORTANCE Hantaviruses have attracted worldwide attention as archetypal emerging pathogens. Recently, increasing evidence has highlighted long noncoding RNAs lncRNAs as key regulators of innate immunity; however, their roles in hantavirus infection remain unknown. In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling.","In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling. This lncRNA was induced by HTNV through the RIG-I–IRF7 pathway in a time- and dose-dependent manner and promoted HTNV-induced IFN production by facilitating RIG-I and DDX60 expression. Intriguingly, NEAT1 relocated SFPQ and formed paraspeckles after HTNV infection, which might reverse inhibitive effects of SFPQ on the transcription of RIG-I and DDX60. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections.","To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections. Text: glycoprotein GP , and viral RNA-dependent polymerase protein RdRp , respectively. Humans become infected by inhaling contaminated aerosols or by coming into contact with rodent excreta, and they develop two severe acute diseases, namely, hemorrhagic fever with renal syndrome HFRS and hantavirus pulmonary syndrome HPS . . Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . .",". Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . . Chinese HFRS cases, mainly caused by Hantaan virus HTNV infection, account for approximately 90% of all global cases, with a mortality rate ranging from 0.1 to 15% . . Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions.","Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions. Cellular pathogen recognition receptors PRRs , including Toll-like receptors TLRs and RIG-I like receptors RLRs , can detect distinct pathogen-associated molecular patterns PAMPs and trigger the expression of IFNs and cytokines. RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression.","RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression. lncRNA nuclear paraspeckle assembly transcript 1 NEAT1 is an essential architectural constituent of paraspeckles in the mammalian nucleus, interacting with Drosophila DBHS RNA-binding proteins such as the splicing factor proline-and glutamine-rich protein SFPQ and the non-POU domain-containing, octamer-binding protein NONO/p54 . . To date, two isoform transcripts of the NEAT1 gene have been identified, namely, the 3.7-kb NEAT1-1 MEN and the 23-kb NEAT1-2 MEN Fig. 1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . .","1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . . , promoting cancer formation in mice by dampening oncogene-dependent activation of p53 . . Nevertheless, studies assessing the function of NEAT1 in viral infections are scarce. Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication.","Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication. Further investigation showed that NEAT1 promoted RIG-I and DDX60 expression by relocating SFPQ and removing the transcriptional inhibitory effects of SFPQ, which are critical for IFN responses against HTNV infection. We also found that RIG-I signaling, rather than TLR3 and TLR4, accounted for the elevation of HTNV-induced NEAT1. Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection.","Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection. As shown in Fig. 1B , the NEAT1 level in the HTNV group was higher than that in the mock group P ϭ 6.86 ϫ 10 Ϫ13 , false discovery rate FDR ϭ 9.75 ϫ 10 Ϫ12 or the 60 Co-inactivated HTNV group P ϭ 1.75 ϫ 10 Ϫ14 , FDR ϭ 3.10 ϫ 10 Ϫ13 ; however, the difference between the 60 Co-inactivated HTNV group and the mock group was not significant P ϭ 0.21034, FDR ϭ 0.58211 . To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig.","To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig. 1A , were applied to quantify NEAT1 RNA isoforms by quantitative real-time PCR qRT-PCR . It has been reported that NEAT1-2 rather than NEAT1-1 plays a key regulatory role in paraspeckle formation . , and we also found that elevated NEAT1 levels depend on live HTNV infection rather than 60 Co-inactivated HTNV stimulation Fig. 1C . Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D .","Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D . To further investigate whether NEAT1 expression was altered in other cell lines, HEK293, HeLa, and A549 cells were used. All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G .","All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G . Similar to the data obtained from HUVECs, NEAT1 was indeed upregulated by HTNV at a multiplicity of infection MOI of 1 beginning at 24 hpi in HUVECs and A549, HEK293, and HeLa cells, and the increasing tendency occurred in a time-dependent manner Fig. 1H . Of note, the NEAT1 elevation at 2 hpi might have been unrelated to the virus but resulted in cellular stress responses. Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I .","Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I . NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. The abovedescribed data showed that HTNV infection increased NEAT1, and we wondered how NEAT1 could reciprocally influence HTNV replication. The small interfering RNA siRNA transfection efficiency in HUVECs was confirmed by flow cytometry, and NEAT1 expression was significantly decreased, as assessed by qRT-PCR after RNA interference RNAi Fig. 2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2.","2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2. Compared with the cells transfected with control siRNA negative control NC , HUVECs with si-NEAT1 could dramatically promote HTNV NP production, and NP expression seemed to be related to the amount of applied si-NEAT1 Fig. 2B . Intriguingly, depletion of NEAT1-2 alone could mimic the antiviral effects of simultaneous NEAT1-1 and NEAT1-2 silencing Fig. 2C , indicating that NEAT1-2 was critical for the antiviral responses. Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing.","Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing. On the other hand, plasmids, of which pCMV-NEAT1-1 is transcribed into the 3.7-kb NEAT1-1 MEN and pCMV-NEAT1-2 is transcribed into the 2-to 3-kb NEAT1-2 MEN , were applied to directly investigate the role of NEAT1 in HTNV infection Fig. 2F . Surprisingly, we found NEAT1-1 overexpression restricted NEAT1-2 transcription Fig. 2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G .","2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G . Furthermore, overexpression of NEAT1-2 instead of NEAT1-1 could efficiently suppress HTNV replication Fig. 2H . NEAT1-1 upregulation even aggravated HTNV infection Fig. 2H , which may be the result of downregulation of NEAT1-2. Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig.","Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig. 2J were limited in HUVECs in which NEAT1-2 was ectopically expressed in comparison to those transfected with control vector or pCMV-NEAT1-1. These data further showed that NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. Alteration of NEAT1-2 affects HTNV-induced IFN expression in HUVECs. IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . .","IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . . It has been reported that the GnT of hantaviruses suppressed IFN- expression of host cells at an early stage of infection . . Here, we also found that HUVECs could not efficiently produce IFN- until 12 hpi at an MOI of 0.1 or until 24 hpi at an MOI of 1 Fig. 3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig.","3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig. 3B , suggesting that NEAT1-2 expression increased just before IFN production. We found that expression of endogenous IFN- mRNA was much lower in cells transfected with si-NEAT1-2 at MOIs of both 0.1 Fig. 3C and 1 Fig. 3D than in those transfected with control siRNA NC . In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E .","In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E . More importantly, HUVECs transfected with pCMV-NEAT1-2 conspicuously increased IFN- gene expression compared with those cells with vector plasmids at 12 hpi MOI ϭ 1 , demonstrating that NEAT1-2 overexpression accelerated robust IFN responses in host cells against HTNV infection. With a dual luciferase reporter system Twenty-four hours after transfection, the cells expressing FAM were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1.","Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the NC group . NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g .","NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 0.1 for 48 h. The expression of HTNV NP was measured by Western blotting. C HUVECs were treated as described for panel A, right, but at an MOI of 0.1. In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.","In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NC group . D HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group .","The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . E HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g .","Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g . Twenty-four hours after transfection, the cells expressing green fluorescent protein GFP were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with control plasmids vector , pCMV-NEAT1-1, or pCMV-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig.","Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig. 3F . These results showed that NEAT1-2 regulated HTNV-induced IFN- expression. To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig.","To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig. 3G . In addition, in cells with high NEAT1-2 expression, treatment with neutralizing antibodies NAbs of IFN-␣ and IFN- could counteract the antiviral effects of NEAT1-2 MOI ϭ 1 , and the compensatory effects were dependent on the magnitude of the NAbs. Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production.","Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production. PRRs maintain a vital role in the promotion of IFN responses, and we conjectured that NEAT1 might amplify IFN responses by modulating these molecules. TLR3, TLR4, and RIG-I have been shown to recognize HTNV infection . . DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate .",". DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate . , Here, we found that multiple Toll-like receptors like TLR1, TLR2, TLR3, and TLR4, as well as MDA5, were increased after HTNV infection, but none of them were influenced by silencing NEAT1-2 Fig. 4A . The upregulated RIG-I and DDX60 were blocked in the cells with low NEAT1-2 expression after HTNV infection Fig. 4A . HUVECs with declining NEAT1-2 expression showed gradually decreasing expression of RIG-I and DDX60 Fig. 4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C .","4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C . These data indicated that NEAT1-2 could positively modulate RIG-I and DDX60 expression, while the role of RIG-I and DDX60 upon HTNV infection is obscure. We then found that RIG-I and DDX60 colocalized after HTNV infection Fig. 4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown .","4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown . Simultaneously knocking down RIG-I and DDX60 significantly promoted HTNV NP expression Fig. 4E , and knockdown of both of them could greatly affect IFN- expression Fig. 4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig.","4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig. 4H , indicating that efficient anti-HTNV responses might depend on the interactive effects of DDX60 and RIG-I. More importantly, RIG-I or/and DDX60 overexpression enhanced HTNV-induced IFN- expression, and they had synergistic effects on IFN- production Fig. 4I and J . Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60.","Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60. NEAT1 was found to interact with SFPQ by RNA immunoprecipitation RIP after HTNV infection Fig. 5A , indicating that modulatory effects of NEAT1 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively .","Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1 for 48 h. The expression of HTNV NP was measured by Western blotting. H HUVECs were treated as described for panel F, right. In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.","In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . H HUVECs were treated as described for panel F, right. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group .","The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . I HUVECs were treated as described for panel F, right. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ.","Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ. Interestingly, the protein level of SFPQ, as well as another paraspeckle-forming constituent, NONO, remained unchanged after HTNV infection Fig. 5B or after NEAT1 overexpression and knockdown Fig. 5C . However, SFPQ became centralized rather than diffuse in the nucleus after HTNV infection Fig. 5D . The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig.","The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig. 5F and G , which might have been related to the increase in RIG-I Fig. 5H and DDX60 Fig. 5I . SFPQ has been suggested to bind to the promoter region of RIG-I and DDX60 ., thus preventing the expression of RIG-I and DDX60. Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription.","Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription. Interestingly, overexpression of the S or M segment of HTNV in HEK293 cells failed to induce NEAT1 expression, suggesting that NEAT1 transcription was closely related to live viral replication Fig. 6A . Of note, the upregulation of NEAT1 by HTNV could not be reversed by applying IFN-I neutralizing antibodies Fig. 6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig.","6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig. 6C, D, and E or cytokines Fig. 6F and G . We conjectured that NEAT1 expression was related to the activation of PRRs. By knocking down several PRRs, we found that the RIG-I and TLR4 pathways played important roles in HTNV-induced NEAT1 upregulation Fig. 7A . Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C .","Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C . Moreover, using STAT1 as a positive control, we found that the transcription factor IRF7, rather than IRF3 and p65, translocated into the nucleus in HTNV-infected HUVECs at 2 dpi Fig. 7D . Furthermore, IRF7 knockdown blocked HTNV-induced NEAT1 upregulation Fig. 7E . Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice.","Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice. Although cell-based experiments revealed that NEAT1-2 is a crucial regulator of innate antihantaviral responses, its function in vivo has remained unclear. To address this question, we intravenously injected siRNAs targeting mouse NEAT1-2 at 1 day before HTNV infection. NEAT1-2 expression levels in the liver, kidney, and spleen were reduced at 2 dpi Fig. 8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions.","8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions. Body weight loss in NEAT1-2-depleted mice was observed from 2 dpi to 5 dpi, and the IFN production in serum was remarkably decreased in the NEAT1-2 silenced group than those in the NC group at 3 dpi Fig. 8B . As expected, NEAT1-2 knockdown mice showed considerably higher HTNV NP levels in the liver, spleen, and kidney at 3 dpi Fig. 8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D .","8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D . In addition, reduced inflammatory cell filtration but increased tissue injury was found in NEAT1-2 knockdown mice during the early stage of infection Fig. 8E . Infiltration of macrophages in the spleen was attenuated Fig. 8F , and the activation of macrophages was also suppressed by flow cytometry; data not shown . Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G .","Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G . Nevertheless, NEAT1-2 silencing had no effect on the production of neutralizing antibodies at 7 dpi data not shown . The above-described findings indicated that NEAT1-2 depletion might influence multiple aspects of the innate immune response in HTNV-infected mice. Innate immunity is a phylogenetically ancient and conserved system that counteracts invading microbes, the regulatory mechanism of which is sophisticated and complex. Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . .","Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . . In this report, we first demonstrated that NEAT1 was induced by HTNV through the RIG-I-IRF7 pathway and served as positive feedback for RIG-I signaling. Using DGE analysis, we observed upregulated NEAT1 and confirmed its alteration in To further determine the function of NEAT1 after HTNV infection in vivo, mice were injected intravenously with si-NEAT1-2 1 g/g or nontarget control siRNA NC 1 g/g ; 1 day later, they were infected with HTNV 100 LD 50 by intramuscular injection. A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group .","A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group . B The effects of NEAT1 on HTNV virulence in mice were determined by body weight loss from 0 to 10 dpi left panel, n ϭ 10 in each group . The IFN- in sera of different groups was measured by ELISA at 3dpi right panel, n ϭ 8 in each group . Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi.","Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi. Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . D NEAT1 effects on HTNV infection kinetics at 3 dpi were determined by testing the HTNV titers in livers, spleens, and kidneys. Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group .","Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group . After HTNV infection, livers in the NC group showed inflammatory cell infiltration in certain regions, while those in the si-NEAT1-2 group showed slight acute viral hepatitis. Spleens in the NC group showed lymph node hyperplasia, while those in the si-NEAT1-2 group were severely congestive. Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented.","Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented. G CD3 ϩ CD8 ϩ IFN-␥ ϩ T cells were analyzed by flow cytometry at 3 dpi, and the results obtained for three mice in each group are presented. different cell lines. To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression.","To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression. By virtue of RNAi, the RIG-I-IRF7 pathway was confirmed to be necessary for HTNV-triggered NEAT1 elevation. Recently, large-scale transcriptomic studies identified numerous noncoding transcripts in the mammalian genome, which were speculated to influence diverse biological processes. Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression .","Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression . . TLR2 activation or tumor necrosis factor alpha TNF-␣ stimulation induces transcription of the lncRNA THRIL, the downregulation of which impairs TNF-␣ and IL-6 secretion . . TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . .",". TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . . The lncRNA Lethe, triggered by TNF-␣ and IL-1, acts as a negative feedback regulator of NF-B signaling . . The roles of lncRNAs in host-virus interactions have been progressively unveiled. Various viruses, such as influenza virus IAV , coronavirus, enterovirus, human immunodeficiency virus HIV , hepatitis B virus HBV , hepatitis C virus HCV , Japanese encephalitis virus JEV , and rabies virus, have been reported to activate the transcription of different lncRNAs in host cells 11, . . . .",". . . Importantly, multiple lncRNAs have been shown to affect the IFN response in recent years and have gradually become hot spots in the field of antiviral research. NeST was shown to enhance IFN-␥ production, controlling the susceptibility of mice to persistent Theiler's virus infection as well as resistance to Salmonella enterica serovar Typhimurium infection . . Both CMPK2 and NRAV were identified as negative regulators of IFN immune reactions. CMPK2, induced by IFN-␣ or HCV infection, suppresses various ISGs, the knockdown of which dramatically blocks HCV replication . . NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . .",". NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . . Numerous lncRNAs, including lnc-ISG15 and ISR2, respond to IFNs such as ISGs, although their actual function requires further investigation . . Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection.","Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection. NEAT1 has been reported to interact with Drosophila DBHS RNA-binding proteins e.g., SFPQ, NONO-p54nrb, and PSPC1 , recruiting them to paraspeckles, a nuclear substructure found in all cultured and primary cells except embryonic stem cells . . The versatile function of NEAT1 is rapidly progressing in multiple areas of biology. NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . .","NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . . NEAT1 also participates in neurodegenerative diseases such as Huntington's disease . and seems to potentially contribute to the elevated production of a number of cytokines and chemokines in patients with systemic lupus erythematosus SLE . . Furthermore, poly I·C can activate NEAT1 transcription through the TLR3 pathway, whereas NEAT1 positively regulates IL-8 transcription and potentially affects the expression of multiple ISGs after poly I·C stimulation . . In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . .","In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . . However, the role of NEAT1 in hantaviral infection remains unclear. In this report, NEAT1 has been identified as an important regulator of the host innate immune system against HTNV infection. Elevated NEAT1 promotes IFN secretion, most likely by enhancing RIG-I and DDX60 expression. DDX60, a DEXD/H box RNA helicase similar to Saccharomyces cerevisiae Ski2, is induced after viral infection . . DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response.",". DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response. However, the antiviral effects of DDX60 seem to vary among viruses . . We found that NEAT1-regulated DDX60 was involved in IFN production in response to HTNV infection. In HTNV-infected cells, double-stranded RNA dsRNA could not be detected, and it is unclear how host PRRs, especially RIG-I, recognize HTNV invasion . . Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation.",". Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation. Of note, we applied multiple cell lines to explore the role of NEAT1 during HTNV infection. HTNV primarily targets vascular endothelial cells in vivo and contributes to the increased vascular permeability and coagulation disorders in HFRS; hence, HUVECs are the most common in vitro cell model to study host innate immunity against HTNV infection or viral pathogenesis . . EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies .",". EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies . . A549 cells were once used to isolate HTNV, and they were confirmed to be a mature model of infection . . . . . Additionally, Huh 7.0 and Huh 7.5 RIG-I Ϫ cells used in our study have been reported to be infected by HTNV by Lee et al. . and can be used as a cell model to study immune responses against HTNV replication . . Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV.",". Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV. Using qRT-PCR, Western blotting, and immunofluorescence assays, we have also shown that both HEK293 and HeLa cells can be infected by HTNV. To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production.","To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production. Alterations in the relative fluorescence intensity of NP after silencing or overexpressing NEAT1-2 did not seem to be as remarkable as qRT-PCR or Western blot analysis results. The NP spotted and exhibited in the ICW results forms obvious stains that mimic PFU. However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays.","However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays. The RNAi studies in vivo are encouraging Fig. 8 , but the NC used by our group was not mutated si-NEAT1-2 i.e., same sense strand, but with a point mutation in the targeting strand . The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling.","The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling. After observing that NEAT1 can regulate IFN expression by HTNV infection, we were interested in the function of NEAT1. We noticed that silencing NEAT1-2 or ectopically expressing NEAT1-2 could not inhibit or enhance IFN expression without HTNV infection Fig. 3F , which indicated that NEAT1-2 could not directly affect IFN- expression. This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression.","This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression. Recent findings have shown that RIG-I signaling is essential for an efficient polyfunctional T cell response during IAV infection . . Indeed, we found that the function of T cells was suppressed after NEAT1-2 depletion in our animal experiments Fig. 8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection.","8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection. HTNV infection induced NEAT1 expression through the RIG-I-IRF7 pathway, while NEAT1 displayed positive feedback for RIG-I signaling. NEAT1 relocated SFPQ from the potential promoter region of several antiviral genes to the paraspeckles, removing the transcriptional inhibitory effects of SFPQ. This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 .","This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 . nontarget control i.e., negative control NC or targeted RIG-I and DDX60 were designed by Gene-Pharma as follows: NC, 5=- UUCUUCGAACGUGUCACGUTT-3=; si-RIG-I-1, 5=-GCCCAUUUAAACCAAGAAATT-3=, si-RIG-I-2, 5=-GGUGGAGGAUAUUUGAACUTT-3=, and si-RIG-I-3, 5=-CCCAACGAUAUCAUUUCUTT-3=; si-DDX60-1, 5=-GUCCAGGUGUCAGUUUGAUTT-3=, si-DDX60-2, 5=-CCGAAGUGAAGAAGGUAAATT-3=, and si-DDX60-3, 5=-GAUGGAUGCUAGGAAAUAUTT-3=. The pCMV-NEAT1-1 and pCMV-NEAT1-2 plasmids, which transcribe NEAT1-1 and NEAT1-2, respectively, were provided by Nakagawa Shinichi . . The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . .",". The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . . Abs against RIG-I, IRF3, IRF7, p65, and STAT1, as well as the neutralizing antibodies against IFN-␣ and IFN-, were purchased from Abcam Cambridge, MA, USA . Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China .","Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China . The Abs targeting CD11b, F4/80, CD3, CD8, IFN-␥, inducible nitric oxide synthase iNOS , and CD206 for flow cytometry were purchased from BD Biosciences San Jose, CA, USA . IFN-␣, -, and -␥, TNF-␣, and IL-1 were from PeperoTech Rocky Hill, NJ . ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis.","ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis. Total cellular RNAs were extracted with RNAiso TaKaRa, Dalian, China , the concentration of which was measured using a NanoDrop 1000 spectrophotometer. Reverse transcription RT was then performed with PrimeScript RT master mix TaKaRa according to the instructions provided by the manufacturer. Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed.","Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed. The qRT-PCR primer sequences for NEAT1, NEAT1-2, IFN-, HTNV S segment, RIG-I, DDX60, -actin, and GAPDH were obtained from previous reports . . The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing.",". The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing. HUVECs with a confluence of 80% in 6 wells were mock infected or infected with live or 60 Co-inactivated HTNV at an MOI of 1. RNAs were extracted as previously described at 24 hpi, and the quality was analyzed using FastQC software by the Beijing Genomics Institute BGI, Shenzhen, China . Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded.","Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded. Tag counts were normalized to TPM transcripts per million by dividing the raw tag count by the total number of tags from each library and multiplying by 1 million. To avoid the possible noise signal from high-throughput sequencing, the genes with average TPM of less than 1 in these three states were excluded. In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01.","In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01. Genes were selected as differentially expressed using a P value threshold of 0.01. FISH and immunofluorescence assays IFA . Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature.","Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature. Prehybridization was performed with lncRNA FISH probe mix at 37°C for 30 min, and then hybridization was performed by adding NEAT1-2 FISH probe mix and incubating the mixture at 37°C overnight. After washing with 4ϫ, 2ϫ, and 1ϫ SSC, the cell nuclei were stained with DAPI 4=,6-diamidino-2-phenylindole . Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently.","Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently. The cells were fixed with 4% PFA for 10 min and permeabilized with 0.1% Triton X-100 for 15 min. Primary Abs were added and incubated at 37°C for 2 h. After five washes with DPBS, secondary Cy3-or fluorescein isothiocyanate FITC -conjugated goat anti-rabbit or goat anti-mouse IgG Sangon, Shanghai, China was added and incubated at 37°C for 2 h. Cell nuclei were stained with DAPI. Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue .","Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue . The samples were then boiled at 95°C for 10 min. The lysates were resolved by 10%, 12%, or 15% SDS-PAGE and transferred to polyvinylidene fluoride PVDF membranes Millipore . The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA .","The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA . ICW assay. The In-Cell Western ICW assay was performed using an Odyssey imaging system Li-Cor according to the manufacturer's instructions. HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1.","HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1. At 48 hpi, HUVECs were washed twice with ice-cold DPBS, fixed with 4% PFA for 10 min, and permeabilized with 1.0% Triton X-100 for 15 min. Cells were added with Li-Cor Odyssey blocking solution at room temperature for 30 min and incubated at 4°C overnight with mouse IgG MAb 1A8 against HTNV NP together with rabbit IgG antibody against -actin, both of which were diluted in PBS containing 3% bovine serum albumin BSA; HyClone . Subsequently, the cells were washed and stained with goat anti-mouse IgG IRDye 800 antibody 1:5,000; Li-Cor and goat anti-rabbit"],"string":"[\n \"Hantavirus infection, which causes zoonotic diseases with a high mortality rate in humans, has long been a global public health concern. Over the past decades, accumulating evidence suggests that long noncoding RNAs lncRNAs play key regulatory roles in innate immunity. However, the involvement of host lncRNAs in hantaviral control remains uncharacterized. In this study, we identified the lncRNA NEAT1 as a vital antiviral modulator. NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection.\",\n \"NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection. Further investigation suggested that NEAT1 served as positive feedback for RIG-I signaling. HTNV infection activated NEAT1 transcription through the RIG-I–IRF7 pathway, whereas NEAT1 removed the transcriptional inhibitory effects of the splicing factor proline- and glutamine-rich protein SFPQ by relocating SFPQ to paraspeckles, thus promoting the expression of RIG-I and DDX60. RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections.\",\n \"RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections. IMPORTANCE Hantaviruses have attracted worldwide attention as archetypal emerging pathogens. Recently, increasing evidence has highlighted long noncoding RNAs lncRNAs as key regulators of innate immunity; however, their roles in hantavirus infection remain unknown. In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling.\",\n \"In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling. This lncRNA was induced by HTNV through the RIG-I–IRF7 pathway in a time- and dose-dependent manner and promoted HTNV-induced IFN production by facilitating RIG-I and DDX60 expression. Intriguingly, NEAT1 relocated SFPQ and formed paraspeckles after HTNV infection, which might reverse inhibitive effects of SFPQ on the transcription of RIG-I and DDX60. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections.\",\n \"To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections. Text: glycoprotein GP , and viral RNA-dependent polymerase protein RdRp , respectively. Humans become infected by inhaling contaminated aerosols or by coming into contact with rodent excreta, and they develop two severe acute diseases, namely, hemorrhagic fever with renal syndrome HFRS and hantavirus pulmonary syndrome HPS . . Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . .\",\n \". Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . . Chinese HFRS cases, mainly caused by Hantaan virus HTNV infection, account for approximately 90% of all global cases, with a mortality rate ranging from 0.1 to 15% . . Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions.\",\n \"Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions. Cellular pathogen recognition receptors PRRs , including Toll-like receptors TLRs and RIG-I like receptors RLRs , can detect distinct pathogen-associated molecular patterns PAMPs and trigger the expression of IFNs and cytokines. RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression.\",\n \"RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression. lncRNA nuclear paraspeckle assembly transcript 1 NEAT1 is an essential architectural constituent of paraspeckles in the mammalian nucleus, interacting with Drosophila DBHS RNA-binding proteins such as the splicing factor proline-and glutamine-rich protein SFPQ and the non-POU domain-containing, octamer-binding protein NONO/p54 . . To date, two isoform transcripts of the NEAT1 gene have been identified, namely, the 3.7-kb NEAT1-1 MEN and the 23-kb NEAT1-2 MEN Fig. 1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . .\",\n \"1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . . , promoting cancer formation in mice by dampening oncogene-dependent activation of p53 . . Nevertheless, studies assessing the function of NEAT1 in viral infections are scarce. Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication.\",\n \"Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication. Further investigation showed that NEAT1 promoted RIG-I and DDX60 expression by relocating SFPQ and removing the transcriptional inhibitory effects of SFPQ, which are critical for IFN responses against HTNV infection. We also found that RIG-I signaling, rather than TLR3 and TLR4, accounted for the elevation of HTNV-induced NEAT1. Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection.\",\n \"Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection. As shown in Fig. 1B , the NEAT1 level in the HTNV group was higher than that in the mock group P ϭ 6.86 ϫ 10 Ϫ13 , false discovery rate FDR ϭ 9.75 ϫ 10 Ϫ12 or the 60 Co-inactivated HTNV group P ϭ 1.75 ϫ 10 Ϫ14 , FDR ϭ 3.10 ϫ 10 Ϫ13 ; however, the difference between the 60 Co-inactivated HTNV group and the mock group was not significant P ϭ 0.21034, FDR ϭ 0.58211 . To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig.\",\n \"To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig. 1A , were applied to quantify NEAT1 RNA isoforms by quantitative real-time PCR qRT-PCR . It has been reported that NEAT1-2 rather than NEAT1-1 plays a key regulatory role in paraspeckle formation . , and we also found that elevated NEAT1 levels depend on live HTNV infection rather than 60 Co-inactivated HTNV stimulation Fig. 1C . Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D .\",\n \"Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D . To further investigate whether NEAT1 expression was altered in other cell lines, HEK293, HeLa, and A549 cells were used. All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G .\",\n \"All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G . Similar to the data obtained from HUVECs, NEAT1 was indeed upregulated by HTNV at a multiplicity of infection MOI of 1 beginning at 24 hpi in HUVECs and A549, HEK293, and HeLa cells, and the increasing tendency occurred in a time-dependent manner Fig. 1H . Of note, the NEAT1 elevation at 2 hpi might have been unrelated to the virus but resulted in cellular stress responses. Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I .\",\n \"Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I . NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. The abovedescribed data showed that HTNV infection increased NEAT1, and we wondered how NEAT1 could reciprocally influence HTNV replication. The small interfering RNA siRNA transfection efficiency in HUVECs was confirmed by flow cytometry, and NEAT1 expression was significantly decreased, as assessed by qRT-PCR after RNA interference RNAi Fig. 2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2.\",\n \"2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2. Compared with the cells transfected with control siRNA negative control NC , HUVECs with si-NEAT1 could dramatically promote HTNV NP production, and NP expression seemed to be related to the amount of applied si-NEAT1 Fig. 2B . Intriguingly, depletion of NEAT1-2 alone could mimic the antiviral effects of simultaneous NEAT1-1 and NEAT1-2 silencing Fig. 2C , indicating that NEAT1-2 was critical for the antiviral responses. Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing.\",\n \"Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing. On the other hand, plasmids, of which pCMV-NEAT1-1 is transcribed into the 3.7-kb NEAT1-1 MEN and pCMV-NEAT1-2 is transcribed into the 2-to 3-kb NEAT1-2 MEN , were applied to directly investigate the role of NEAT1 in HTNV infection Fig. 2F . Surprisingly, we found NEAT1-1 overexpression restricted NEAT1-2 transcription Fig. 2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G .\",\n \"2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G . Furthermore, overexpression of NEAT1-2 instead of NEAT1-1 could efficiently suppress HTNV replication Fig. 2H . NEAT1-1 upregulation even aggravated HTNV infection Fig. 2H , which may be the result of downregulation of NEAT1-2. Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig.\",\n \"Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig. 2J were limited in HUVECs in which NEAT1-2 was ectopically expressed in comparison to those transfected with control vector or pCMV-NEAT1-1. These data further showed that NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. Alteration of NEAT1-2 affects HTNV-induced IFN expression in HUVECs. IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . .\",\n \"IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . . It has been reported that the GnT of hantaviruses suppressed IFN- expression of host cells at an early stage of infection . . Here, we also found that HUVECs could not efficiently produce IFN- until 12 hpi at an MOI of 0.1 or until 24 hpi at an MOI of 1 Fig. 3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig.\",\n \"3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig. 3B , suggesting that NEAT1-2 expression increased just before IFN production. We found that expression of endogenous IFN- mRNA was much lower in cells transfected with si-NEAT1-2 at MOIs of both 0.1 Fig. 3C and 1 Fig. 3D than in those transfected with control siRNA NC . In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E .\",\n \"In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E . More importantly, HUVECs transfected with pCMV-NEAT1-2 conspicuously increased IFN- gene expression compared with those cells with vector plasmids at 12 hpi MOI ϭ 1 , demonstrating that NEAT1-2 overexpression accelerated robust IFN responses in host cells against HTNV infection. With a dual luciferase reporter system Twenty-four hours after transfection, the cells expressing FAM were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1.\",\n \"Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the NC group . NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g .\",\n \"NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 0.1 for 48 h. The expression of HTNV NP was measured by Western blotting. C HUVECs were treated as described for panel A, right, but at an MOI of 0.1. In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.\",\n \"In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NC group . D HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group .\",\n \"The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . E HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g .\",\n \"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g . Twenty-four hours after transfection, the cells expressing green fluorescent protein GFP were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with control plasmids vector , pCMV-NEAT1-1, or pCMV-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig.\",\n \"Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig. 3F . These results showed that NEAT1-2 regulated HTNV-induced IFN- expression. To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig.\",\n \"To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig. 3G . In addition, in cells with high NEAT1-2 expression, treatment with neutralizing antibodies NAbs of IFN-␣ and IFN- could counteract the antiviral effects of NEAT1-2 MOI ϭ 1 , and the compensatory effects were dependent on the magnitude of the NAbs. Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production.\",\n \"Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production. PRRs maintain a vital role in the promotion of IFN responses, and we conjectured that NEAT1 might amplify IFN responses by modulating these molecules. TLR3, TLR4, and RIG-I have been shown to recognize HTNV infection . . DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate .\",\n \". DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate . , Here, we found that multiple Toll-like receptors like TLR1, TLR2, TLR3, and TLR4, as well as MDA5, were increased after HTNV infection, but none of them were influenced by silencing NEAT1-2 Fig. 4A . The upregulated RIG-I and DDX60 were blocked in the cells with low NEAT1-2 expression after HTNV infection Fig. 4A . HUVECs with declining NEAT1-2 expression showed gradually decreasing expression of RIG-I and DDX60 Fig. 4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C .\",\n \"4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C . These data indicated that NEAT1-2 could positively modulate RIG-I and DDX60 expression, while the role of RIG-I and DDX60 upon HTNV infection is obscure. We then found that RIG-I and DDX60 colocalized after HTNV infection Fig. 4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown .\",\n \"4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown . Simultaneously knocking down RIG-I and DDX60 significantly promoted HTNV NP expression Fig. 4E , and knockdown of both of them could greatly affect IFN- expression Fig. 4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig.\",\n \"4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig. 4H , indicating that efficient anti-HTNV responses might depend on the interactive effects of DDX60 and RIG-I. More importantly, RIG-I or/and DDX60 overexpression enhanced HTNV-induced IFN- expression, and they had synergistic effects on IFN- production Fig. 4I and J . Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60.\",\n \"Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60. NEAT1 was found to interact with SFPQ by RNA immunoprecipitation RIP after HTNV infection Fig. 5A , indicating that modulatory effects of NEAT1 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively .\",\n \"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1 for 48 h. The expression of HTNV NP was measured by Western blotting. H HUVECs were treated as described for panel F, right. In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.\",\n \"In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . H HUVECs were treated as described for panel F, right. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group .\",\n \"The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . I HUVECs were treated as described for panel F, right. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ.\",\n \"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ. Interestingly, the protein level of SFPQ, as well as another paraspeckle-forming constituent, NONO, remained unchanged after HTNV infection Fig. 5B or after NEAT1 overexpression and knockdown Fig. 5C . However, SFPQ became centralized rather than diffuse in the nucleus after HTNV infection Fig. 5D . The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig.\",\n \"The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig. 5F and G , which might have been related to the increase in RIG-I Fig. 5H and DDX60 Fig. 5I . SFPQ has been suggested to bind to the promoter region of RIG-I and DDX60 ., thus preventing the expression of RIG-I and DDX60. Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription.\",\n \"Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription. Interestingly, overexpression of the S or M segment of HTNV in HEK293 cells failed to induce NEAT1 expression, suggesting that NEAT1 transcription was closely related to live viral replication Fig. 6A . Of note, the upregulation of NEAT1 by HTNV could not be reversed by applying IFN-I neutralizing antibodies Fig. 6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig.\",\n \"6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig. 6C, D, and E or cytokines Fig. 6F and G . We conjectured that NEAT1 expression was related to the activation of PRRs. By knocking down several PRRs, we found that the RIG-I and TLR4 pathways played important roles in HTNV-induced NEAT1 upregulation Fig. 7A . Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C .\",\n \"Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C . Moreover, using STAT1 as a positive control, we found that the transcription factor IRF7, rather than IRF3 and p65, translocated into the nucleus in HTNV-infected HUVECs at 2 dpi Fig. 7D . Furthermore, IRF7 knockdown blocked HTNV-induced NEAT1 upregulation Fig. 7E . Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice.\",\n \"Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice. Although cell-based experiments revealed that NEAT1-2 is a crucial regulator of innate antihantaviral responses, its function in vivo has remained unclear. To address this question, we intravenously injected siRNAs targeting mouse NEAT1-2 at 1 day before HTNV infection. NEAT1-2 expression levels in the liver, kidney, and spleen were reduced at 2 dpi Fig. 8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions.\",\n \"8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions. Body weight loss in NEAT1-2-depleted mice was observed from 2 dpi to 5 dpi, and the IFN production in serum was remarkably decreased in the NEAT1-2 silenced group than those in the NC group at 3 dpi Fig. 8B . As expected, NEAT1-2 knockdown mice showed considerably higher HTNV NP levels in the liver, spleen, and kidney at 3 dpi Fig. 8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D .\",\n \"8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D . In addition, reduced inflammatory cell filtration but increased tissue injury was found in NEAT1-2 knockdown mice during the early stage of infection Fig. 8E . Infiltration of macrophages in the spleen was attenuated Fig. 8F , and the activation of macrophages was also suppressed by flow cytometry; data not shown . Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G .\",\n \"Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G . Nevertheless, NEAT1-2 silencing had no effect on the production of neutralizing antibodies at 7 dpi data not shown . The above-described findings indicated that NEAT1-2 depletion might influence multiple aspects of the innate immune response in HTNV-infected mice. Innate immunity is a phylogenetically ancient and conserved system that counteracts invading microbes, the regulatory mechanism of which is sophisticated and complex. Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . .\",\n \"Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . . In this report, we first demonstrated that NEAT1 was induced by HTNV through the RIG-I-IRF7 pathway and served as positive feedback for RIG-I signaling. Using DGE analysis, we observed upregulated NEAT1 and confirmed its alteration in To further determine the function of NEAT1 after HTNV infection in vivo, mice were injected intravenously with si-NEAT1-2 1 g/g or nontarget control siRNA NC 1 g/g ; 1 day later, they were infected with HTNV 100 LD 50 by intramuscular injection. A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group .\",\n \"A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group . B The effects of NEAT1 on HTNV virulence in mice were determined by body weight loss from 0 to 10 dpi left panel, n ϭ 10 in each group . The IFN- in sera of different groups was measured by ELISA at 3dpi right panel, n ϭ 8 in each group . Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi.\",\n \"Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi. Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . D NEAT1 effects on HTNV infection kinetics at 3 dpi were determined by testing the HTNV titers in livers, spleens, and kidneys. Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group .\",\n \"Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group . After HTNV infection, livers in the NC group showed inflammatory cell infiltration in certain regions, while those in the si-NEAT1-2 group showed slight acute viral hepatitis. Spleens in the NC group showed lymph node hyperplasia, while those in the si-NEAT1-2 group were severely congestive. Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented.\",\n \"Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented. G CD3 ϩ CD8 ϩ IFN-␥ ϩ T cells were analyzed by flow cytometry at 3 dpi, and the results obtained for three mice in each group are presented. different cell lines. To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression.\",\n \"To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression. By virtue of RNAi, the RIG-I-IRF7 pathway was confirmed to be necessary for HTNV-triggered NEAT1 elevation. Recently, large-scale transcriptomic studies identified numerous noncoding transcripts in the mammalian genome, which were speculated to influence diverse biological processes. Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression .\",\n \"Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression . . TLR2 activation or tumor necrosis factor alpha TNF-␣ stimulation induces transcription of the lncRNA THRIL, the downregulation of which impairs TNF-␣ and IL-6 secretion . . TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . .\",\n \". TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . . The lncRNA Lethe, triggered by TNF-␣ and IL-1, acts as a negative feedback regulator of NF-B signaling . . The roles of lncRNAs in host-virus interactions have been progressively unveiled. Various viruses, such as influenza virus IAV , coronavirus, enterovirus, human immunodeficiency virus HIV , hepatitis B virus HBV , hepatitis C virus HCV , Japanese encephalitis virus JEV , and rabies virus, have been reported to activate the transcription of different lncRNAs in host cells 11, . . . .\",\n \". . . Importantly, multiple lncRNAs have been shown to affect the IFN response in recent years and have gradually become hot spots in the field of antiviral research. NeST was shown to enhance IFN-␥ production, controlling the susceptibility of mice to persistent Theiler's virus infection as well as resistance to Salmonella enterica serovar Typhimurium infection . . Both CMPK2 and NRAV were identified as negative regulators of IFN immune reactions. CMPK2, induced by IFN-␣ or HCV infection, suppresses various ISGs, the knockdown of which dramatically blocks HCV replication . . NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . .\",\n \". NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . . Numerous lncRNAs, including lnc-ISG15 and ISR2, respond to IFNs such as ISGs, although their actual function requires further investigation . . Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection.\",\n \"Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection. NEAT1 has been reported to interact with Drosophila DBHS RNA-binding proteins e.g., SFPQ, NONO-p54nrb, and PSPC1 , recruiting them to paraspeckles, a nuclear substructure found in all cultured and primary cells except embryonic stem cells . . The versatile function of NEAT1 is rapidly progressing in multiple areas of biology. NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . .\",\n \"NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . . NEAT1 also participates in neurodegenerative diseases such as Huntington's disease . and seems to potentially contribute to the elevated production of a number of cytokines and chemokines in patients with systemic lupus erythematosus SLE . . Furthermore, poly I·C can activate NEAT1 transcription through the TLR3 pathway, whereas NEAT1 positively regulates IL-8 transcription and potentially affects the expression of multiple ISGs after poly I·C stimulation . . In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . .\",\n \"In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . . However, the role of NEAT1 in hantaviral infection remains unclear. In this report, NEAT1 has been identified as an important regulator of the host innate immune system against HTNV infection. Elevated NEAT1 promotes IFN secretion, most likely by enhancing RIG-I and DDX60 expression. DDX60, a DEXD/H box RNA helicase similar to Saccharomyces cerevisiae Ski2, is induced after viral infection . . DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response.\",\n \". DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response. However, the antiviral effects of DDX60 seem to vary among viruses . . We found that NEAT1-regulated DDX60 was involved in IFN production in response to HTNV infection. In HTNV-infected cells, double-stranded RNA dsRNA could not be detected, and it is unclear how host PRRs, especially RIG-I, recognize HTNV invasion . . Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation.\",\n \". Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation. Of note, we applied multiple cell lines to explore the role of NEAT1 during HTNV infection. HTNV primarily targets vascular endothelial cells in vivo and contributes to the increased vascular permeability and coagulation disorders in HFRS; hence, HUVECs are the most common in vitro cell model to study host innate immunity against HTNV infection or viral pathogenesis . . EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies .\",\n \". EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies . . A549 cells were once used to isolate HTNV, and they were confirmed to be a mature model of infection . . . . . Additionally, Huh 7.0 and Huh 7.5 RIG-I Ϫ cells used in our study have been reported to be infected by HTNV by Lee et al. . and can be used as a cell model to study immune responses against HTNV replication . . Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV.\",\n \". Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV. Using qRT-PCR, Western blotting, and immunofluorescence assays, we have also shown that both HEK293 and HeLa cells can be infected by HTNV. To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production.\",\n \"To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production. Alterations in the relative fluorescence intensity of NP after silencing or overexpressing NEAT1-2 did not seem to be as remarkable as qRT-PCR or Western blot analysis results. The NP spotted and exhibited in the ICW results forms obvious stains that mimic PFU. However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays.\",\n \"However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays. The RNAi studies in vivo are encouraging Fig. 8 , but the NC used by our group was not mutated si-NEAT1-2 i.e., same sense strand, but with a point mutation in the targeting strand . The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling.\",\n \"The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling. After observing that NEAT1 can regulate IFN expression by HTNV infection, we were interested in the function of NEAT1. We noticed that silencing NEAT1-2 or ectopically expressing NEAT1-2 could not inhibit or enhance IFN expression without HTNV infection Fig. 3F , which indicated that NEAT1-2 could not directly affect IFN- expression. This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression.\",\n \"This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression. Recent findings have shown that RIG-I signaling is essential for an efficient polyfunctional T cell response during IAV infection . . Indeed, we found that the function of T cells was suppressed after NEAT1-2 depletion in our animal experiments Fig. 8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection.\",\n \"8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection. HTNV infection induced NEAT1 expression through the RIG-I-IRF7 pathway, while NEAT1 displayed positive feedback for RIG-I signaling. NEAT1 relocated SFPQ from the potential promoter region of several antiviral genes to the paraspeckles, removing the transcriptional inhibitory effects of SFPQ. This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 .\",\n \"This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 . nontarget control i.e., negative control NC or targeted RIG-I and DDX60 were designed by Gene-Pharma as follows: NC, 5=- UUCUUCGAACGUGUCACGUTT-3=; si-RIG-I-1, 5=-GCCCAUUUAAACCAAGAAATT-3=, si-RIG-I-2, 5=-GGUGGAGGAUAUUUGAACUTT-3=, and si-RIG-I-3, 5=-CCCAACGAUAUCAUUUCUTT-3=; si-DDX60-1, 5=-GUCCAGGUGUCAGUUUGAUTT-3=, si-DDX60-2, 5=-CCGAAGUGAAGAAGGUAAATT-3=, and si-DDX60-3, 5=-GAUGGAUGCUAGGAAAUAUTT-3=. The pCMV-NEAT1-1 and pCMV-NEAT1-2 plasmids, which transcribe NEAT1-1 and NEAT1-2, respectively, were provided by Nakagawa Shinichi . . The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . .\",\n \". The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . . Abs against RIG-I, IRF3, IRF7, p65, and STAT1, as well as the neutralizing antibodies against IFN-␣ and IFN-, were purchased from Abcam Cambridge, MA, USA . Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China .\",\n \"Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China . The Abs targeting CD11b, F4/80, CD3, CD8, IFN-␥, inducible nitric oxide synthase iNOS , and CD206 for flow cytometry were purchased from BD Biosciences San Jose, CA, USA . IFN-␣, -, and -␥, TNF-␣, and IL-1 were from PeperoTech Rocky Hill, NJ . ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis.\",\n \"ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis. Total cellular RNAs were extracted with RNAiso TaKaRa, Dalian, China , the concentration of which was measured using a NanoDrop 1000 spectrophotometer. Reverse transcription RT was then performed with PrimeScript RT master mix TaKaRa according to the instructions provided by the manufacturer. Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed.\",\n \"Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed. The qRT-PCR primer sequences for NEAT1, NEAT1-2, IFN-, HTNV S segment, RIG-I, DDX60, -actin, and GAPDH were obtained from previous reports . . The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing.\",\n \". The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing. HUVECs with a confluence of 80% in 6 wells were mock infected or infected with live or 60 Co-inactivated HTNV at an MOI of 1. RNAs were extracted as previously described at 24 hpi, and the quality was analyzed using FastQC software by the Beijing Genomics Institute BGI, Shenzhen, China . Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded.\",\n \"Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded. Tag counts were normalized to TPM transcripts per million by dividing the raw tag count by the total number of tags from each library and multiplying by 1 million. To avoid the possible noise signal from high-throughput sequencing, the genes with average TPM of less than 1 in these three states were excluded. In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01.\",\n \"In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01. Genes were selected as differentially expressed using a P value threshold of 0.01. FISH and immunofluorescence assays IFA . Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature.\",\n \"Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature. Prehybridization was performed with lncRNA FISH probe mix at 37°C for 30 min, and then hybridization was performed by adding NEAT1-2 FISH probe mix and incubating the mixture at 37°C overnight. After washing with 4ϫ, 2ϫ, and 1ϫ SSC, the cell nuclei were stained with DAPI 4=,6-diamidino-2-phenylindole . Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently.\",\n \"Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently. The cells were fixed with 4% PFA for 10 min and permeabilized with 0.1% Triton X-100 for 15 min. Primary Abs were added and incubated at 37°C for 2 h. After five washes with DPBS, secondary Cy3-or fluorescein isothiocyanate FITC -conjugated goat anti-rabbit or goat anti-mouse IgG Sangon, Shanghai, China was added and incubated at 37°C for 2 h. Cell nuclei were stained with DAPI. Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue .\",\n \"Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue . The samples were then boiled at 95°C for 10 min. The lysates were resolved by 10%, 12%, or 15% SDS-PAGE and transferred to polyvinylidene fluoride PVDF membranes Millipore . The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA .\",\n \"The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA . ICW assay. The In-Cell Western ICW assay was performed using an Odyssey imaging system Li-Cor according to the manufacturer's instructions. HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1.\",\n \"HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1. At 48 hpi, HUVECs were washed twice with ice-cold DPBS, fixed with 4% PFA for 10 min, and permeabilized with 1.0% Triton X-100 for 15 min. Cells were added with Li-Cor Odyssey blocking solution at room temperature for 30 min and incubated at 4°C overnight with mouse IgG MAb 1A8 against HTNV NP together with rabbit IgG antibody against -actin, both of which were diluted in PBS containing 3% bovine serum albumin BSA; HyClone . Subsequently, the cells were washed and stained with goat anti-mouse IgG IRDye 800 antibody 1:5,000; Li-Cor and goat anti-rabbit\"\n]"},"document_id":{"kind":"number","value":2652,"string":"2,652"},"id":{"kind":"number","value":5312,"string":"5,312"}}},{"rowIdx":673,"cells":{"question":{"kind":"string","value":"What was the major contribution of this study?"},"answer":{"kind":"string","value":"the first study to describe the role of NEAT1 in HTNV infection"},"context_chunks":{"kind":"list like","value":["Hantavirus infection, which causes zoonotic diseases with a high mortality rate in humans, has long been a global public health concern. Over the past decades, accumulating evidence suggests that long noncoding RNAs lncRNAs play key regulatory roles in innate immunity. However, the involvement of host lncRNAs in hantaviral control remains uncharacterized. In this study, we identified the lncRNA NEAT1 as a vital antiviral modulator. NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection.","NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection. Further investigation suggested that NEAT1 served as positive feedback for RIG-I signaling. HTNV infection activated NEAT1 transcription through the RIG-I–IRF7 pathway, whereas NEAT1 removed the transcriptional inhibitory effects of the splicing factor proline- and glutamine-rich protein SFPQ by relocating SFPQ to paraspeckles, thus promoting the expression of RIG-I and DDX60. RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections.","RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections. IMPORTANCE Hantaviruses have attracted worldwide attention as archetypal emerging pathogens. Recently, increasing evidence has highlighted long noncoding RNAs lncRNAs as key regulators of innate immunity; however, their roles in hantavirus infection remain unknown. In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling.","In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling. This lncRNA was induced by HTNV through the RIG-I–IRF7 pathway in a time- and dose-dependent manner and promoted HTNV-induced IFN production by facilitating RIG-I and DDX60 expression. Intriguingly, NEAT1 relocated SFPQ and formed paraspeckles after HTNV infection, which might reverse inhibitive effects of SFPQ on the transcription of RIG-I and DDX60. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections.","To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections. Text: glycoprotein GP , and viral RNA-dependent polymerase protein RdRp , respectively. Humans become infected by inhaling contaminated aerosols or by coming into contact with rodent excreta, and they develop two severe acute diseases, namely, hemorrhagic fever with renal syndrome HFRS and hantavirus pulmonary syndrome HPS . . Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . .",". Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . . Chinese HFRS cases, mainly caused by Hantaan virus HTNV infection, account for approximately 90% of all global cases, with a mortality rate ranging from 0.1 to 15% . . Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions.","Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions. Cellular pathogen recognition receptors PRRs , including Toll-like receptors TLRs and RIG-I like receptors RLRs , can detect distinct pathogen-associated molecular patterns PAMPs and trigger the expression of IFNs and cytokines. RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression.","RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression. lncRNA nuclear paraspeckle assembly transcript 1 NEAT1 is an essential architectural constituent of paraspeckles in the mammalian nucleus, interacting with Drosophila DBHS RNA-binding proteins such as the splicing factor proline-and glutamine-rich protein SFPQ and the non-POU domain-containing, octamer-binding protein NONO/p54 . . To date, two isoform transcripts of the NEAT1 gene have been identified, namely, the 3.7-kb NEAT1-1 MEN and the 23-kb NEAT1-2 MEN Fig. 1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . .","1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . . , promoting cancer formation in mice by dampening oncogene-dependent activation of p53 . . Nevertheless, studies assessing the function of NEAT1 in viral infections are scarce. Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication.","Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication. Further investigation showed that NEAT1 promoted RIG-I and DDX60 expression by relocating SFPQ and removing the transcriptional inhibitory effects of SFPQ, which are critical for IFN responses against HTNV infection. We also found that RIG-I signaling, rather than TLR3 and TLR4, accounted for the elevation of HTNV-induced NEAT1. Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection.","Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection. As shown in Fig. 1B , the NEAT1 level in the HTNV group was higher than that in the mock group P ϭ 6.86 ϫ 10 Ϫ13 , false discovery rate FDR ϭ 9.75 ϫ 10 Ϫ12 or the 60 Co-inactivated HTNV group P ϭ 1.75 ϫ 10 Ϫ14 , FDR ϭ 3.10 ϫ 10 Ϫ13 ; however, the difference between the 60 Co-inactivated HTNV group and the mock group was not significant P ϭ 0.21034, FDR ϭ 0.58211 . To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig.","To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig. 1A , were applied to quantify NEAT1 RNA isoforms by quantitative real-time PCR qRT-PCR . It has been reported that NEAT1-2 rather than NEAT1-1 plays a key regulatory role in paraspeckle formation . , and we also found that elevated NEAT1 levels depend on live HTNV infection rather than 60 Co-inactivated HTNV stimulation Fig. 1C . Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D .","Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D . To further investigate whether NEAT1 expression was altered in other cell lines, HEK293, HeLa, and A549 cells were used. All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G .","All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G . Similar to the data obtained from HUVECs, NEAT1 was indeed upregulated by HTNV at a multiplicity of infection MOI of 1 beginning at 24 hpi in HUVECs and A549, HEK293, and HeLa cells, and the increasing tendency occurred in a time-dependent manner Fig. 1H . Of note, the NEAT1 elevation at 2 hpi might have been unrelated to the virus but resulted in cellular stress responses. Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I .","Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I . NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. The abovedescribed data showed that HTNV infection increased NEAT1, and we wondered how NEAT1 could reciprocally influence HTNV replication. The small interfering RNA siRNA transfection efficiency in HUVECs was confirmed by flow cytometry, and NEAT1 expression was significantly decreased, as assessed by qRT-PCR after RNA interference RNAi Fig. 2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2.","2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2. Compared with the cells transfected with control siRNA negative control NC , HUVECs with si-NEAT1 could dramatically promote HTNV NP production, and NP expression seemed to be related to the amount of applied si-NEAT1 Fig. 2B . Intriguingly, depletion of NEAT1-2 alone could mimic the antiviral effects of simultaneous NEAT1-1 and NEAT1-2 silencing Fig. 2C , indicating that NEAT1-2 was critical for the antiviral responses. Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing.","Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing. On the other hand, plasmids, of which pCMV-NEAT1-1 is transcribed into the 3.7-kb NEAT1-1 MEN and pCMV-NEAT1-2 is transcribed into the 2-to 3-kb NEAT1-2 MEN , were applied to directly investigate the role of NEAT1 in HTNV infection Fig. 2F . Surprisingly, we found NEAT1-1 overexpression restricted NEAT1-2 transcription Fig. 2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G .","2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G . Furthermore, overexpression of NEAT1-2 instead of NEAT1-1 could efficiently suppress HTNV replication Fig. 2H . NEAT1-1 upregulation even aggravated HTNV infection Fig. 2H , which may be the result of downregulation of NEAT1-2. Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig.","Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig. 2J were limited in HUVECs in which NEAT1-2 was ectopically expressed in comparison to those transfected with control vector or pCMV-NEAT1-1. These data further showed that NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. Alteration of NEAT1-2 affects HTNV-induced IFN expression in HUVECs. IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . .","IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . . It has been reported that the GnT of hantaviruses suppressed IFN- expression of host cells at an early stage of infection . . Here, we also found that HUVECs could not efficiently produce IFN- until 12 hpi at an MOI of 0.1 or until 24 hpi at an MOI of 1 Fig. 3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig.","3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig. 3B , suggesting that NEAT1-2 expression increased just before IFN production. We found that expression of endogenous IFN- mRNA was much lower in cells transfected with si-NEAT1-2 at MOIs of both 0.1 Fig. 3C and 1 Fig. 3D than in those transfected with control siRNA NC . In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E .","In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E . More importantly, HUVECs transfected with pCMV-NEAT1-2 conspicuously increased IFN- gene expression compared with those cells with vector plasmids at 12 hpi MOI ϭ 1 , demonstrating that NEAT1-2 overexpression accelerated robust IFN responses in host cells against HTNV infection. With a dual luciferase reporter system Twenty-four hours after transfection, the cells expressing FAM were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1.","Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the NC group . NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g .","NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 0.1 for 48 h. The expression of HTNV NP was measured by Western blotting. C HUVECs were treated as described for panel A, right, but at an MOI of 0.1. In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.","In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NC group . D HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group .","The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . E HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g .","Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g . Twenty-four hours after transfection, the cells expressing green fluorescent protein GFP were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with control plasmids vector , pCMV-NEAT1-1, or pCMV-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig.","Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig. 3F . These results showed that NEAT1-2 regulated HTNV-induced IFN- expression. To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig.","To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig. 3G . In addition, in cells with high NEAT1-2 expression, treatment with neutralizing antibodies NAbs of IFN-␣ and IFN- could counteract the antiviral effects of NEAT1-2 MOI ϭ 1 , and the compensatory effects were dependent on the magnitude of the NAbs. Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production.","Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production. PRRs maintain a vital role in the promotion of IFN responses, and we conjectured that NEAT1 might amplify IFN responses by modulating these molecules. TLR3, TLR4, and RIG-I have been shown to recognize HTNV infection . . DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate .",". DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate . , Here, we found that multiple Toll-like receptors like TLR1, TLR2, TLR3, and TLR4, as well as MDA5, were increased after HTNV infection, but none of them were influenced by silencing NEAT1-2 Fig. 4A . The upregulated RIG-I and DDX60 were blocked in the cells with low NEAT1-2 expression after HTNV infection Fig. 4A . HUVECs with declining NEAT1-2 expression showed gradually decreasing expression of RIG-I and DDX60 Fig. 4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C .","4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C . These data indicated that NEAT1-2 could positively modulate RIG-I and DDX60 expression, while the role of RIG-I and DDX60 upon HTNV infection is obscure. We then found that RIG-I and DDX60 colocalized after HTNV infection Fig. 4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown .","4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown . Simultaneously knocking down RIG-I and DDX60 significantly promoted HTNV NP expression Fig. 4E , and knockdown of both of them could greatly affect IFN- expression Fig. 4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig.","4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig. 4H , indicating that efficient anti-HTNV responses might depend on the interactive effects of DDX60 and RIG-I. More importantly, RIG-I or/and DDX60 overexpression enhanced HTNV-induced IFN- expression, and they had synergistic effects on IFN- production Fig. 4I and J . Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60.","Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60. NEAT1 was found to interact with SFPQ by RNA immunoprecipitation RIP after HTNV infection Fig. 5A , indicating that modulatory effects of NEAT1 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively .","Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1 for 48 h. The expression of HTNV NP was measured by Western blotting. H HUVECs were treated as described for panel F, right. In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.","In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . H HUVECs were treated as described for panel F, right. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group .","The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . I HUVECs were treated as described for panel F, right. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ.","Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ. Interestingly, the protein level of SFPQ, as well as another paraspeckle-forming constituent, NONO, remained unchanged after HTNV infection Fig. 5B or after NEAT1 overexpression and knockdown Fig. 5C . However, SFPQ became centralized rather than diffuse in the nucleus after HTNV infection Fig. 5D . The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig.","The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig. 5F and G , which might have been related to the increase in RIG-I Fig. 5H and DDX60 Fig. 5I . SFPQ has been suggested to bind to the promoter region of RIG-I and DDX60 ., thus preventing the expression of RIG-I and DDX60. Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription.","Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription. Interestingly, overexpression of the S or M segment of HTNV in HEK293 cells failed to induce NEAT1 expression, suggesting that NEAT1 transcription was closely related to live viral replication Fig. 6A . Of note, the upregulation of NEAT1 by HTNV could not be reversed by applying IFN-I neutralizing antibodies Fig. 6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig.","6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig. 6C, D, and E or cytokines Fig. 6F and G . We conjectured that NEAT1 expression was related to the activation of PRRs. By knocking down several PRRs, we found that the RIG-I and TLR4 pathways played important roles in HTNV-induced NEAT1 upregulation Fig. 7A . Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C .","Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C . Moreover, using STAT1 as a positive control, we found that the transcription factor IRF7, rather than IRF3 and p65, translocated into the nucleus in HTNV-infected HUVECs at 2 dpi Fig. 7D . Furthermore, IRF7 knockdown blocked HTNV-induced NEAT1 upregulation Fig. 7E . Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice.","Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice. Although cell-based experiments revealed that NEAT1-2 is a crucial regulator of innate antihantaviral responses, its function in vivo has remained unclear. To address this question, we intravenously injected siRNAs targeting mouse NEAT1-2 at 1 day before HTNV infection. NEAT1-2 expression levels in the liver, kidney, and spleen were reduced at 2 dpi Fig. 8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions.","8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions. Body weight loss in NEAT1-2-depleted mice was observed from 2 dpi to 5 dpi, and the IFN production in serum was remarkably decreased in the NEAT1-2 silenced group than those in the NC group at 3 dpi Fig. 8B . As expected, NEAT1-2 knockdown mice showed considerably higher HTNV NP levels in the liver, spleen, and kidney at 3 dpi Fig. 8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D .","8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D . In addition, reduced inflammatory cell filtration but increased tissue injury was found in NEAT1-2 knockdown mice during the early stage of infection Fig. 8E . Infiltration of macrophages in the spleen was attenuated Fig. 8F , and the activation of macrophages was also suppressed by flow cytometry; data not shown . Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G .","Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G . Nevertheless, NEAT1-2 silencing had no effect on the production of neutralizing antibodies at 7 dpi data not shown . The above-described findings indicated that NEAT1-2 depletion might influence multiple aspects of the innate immune response in HTNV-infected mice. Innate immunity is a phylogenetically ancient and conserved system that counteracts invading microbes, the regulatory mechanism of which is sophisticated and complex. Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . .","Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . . In this report, we first demonstrated that NEAT1 was induced by HTNV through the RIG-I-IRF7 pathway and served as positive feedback for RIG-I signaling. Using DGE analysis, we observed upregulated NEAT1 and confirmed its alteration in To further determine the function of NEAT1 after HTNV infection in vivo, mice were injected intravenously with si-NEAT1-2 1 g/g or nontarget control siRNA NC 1 g/g ; 1 day later, they were infected with HTNV 100 LD 50 by intramuscular injection. A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group .","A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group . B The effects of NEAT1 on HTNV virulence in mice were determined by body weight loss from 0 to 10 dpi left panel, n ϭ 10 in each group . The IFN- in sera of different groups was measured by ELISA at 3dpi right panel, n ϭ 8 in each group . Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi.","Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi. Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . D NEAT1 effects on HTNV infection kinetics at 3 dpi were determined by testing the HTNV titers in livers, spleens, and kidneys. Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group .","Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group . After HTNV infection, livers in the NC group showed inflammatory cell infiltration in certain regions, while those in the si-NEAT1-2 group showed slight acute viral hepatitis. Spleens in the NC group showed lymph node hyperplasia, while those in the si-NEAT1-2 group were severely congestive. Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented.","Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented. G CD3 ϩ CD8 ϩ IFN-␥ ϩ T cells were analyzed by flow cytometry at 3 dpi, and the results obtained for three mice in each group are presented. different cell lines. To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression.","To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression. By virtue of RNAi, the RIG-I-IRF7 pathway was confirmed to be necessary for HTNV-triggered NEAT1 elevation. Recently, large-scale transcriptomic studies identified numerous noncoding transcripts in the mammalian genome, which were speculated to influence diverse biological processes. Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression .","Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression . . TLR2 activation or tumor necrosis factor alpha TNF-␣ stimulation induces transcription of the lncRNA THRIL, the downregulation of which impairs TNF-␣ and IL-6 secretion . . TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . .",". TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . . The lncRNA Lethe, triggered by TNF-␣ and IL-1, acts as a negative feedback regulator of NF-B signaling . . The roles of lncRNAs in host-virus interactions have been progressively unveiled. Various viruses, such as influenza virus IAV , coronavirus, enterovirus, human immunodeficiency virus HIV , hepatitis B virus HBV , hepatitis C virus HCV , Japanese encephalitis virus JEV , and rabies virus, have been reported to activate the transcription of different lncRNAs in host cells 11, . . . .",". . . Importantly, multiple lncRNAs have been shown to affect the IFN response in recent years and have gradually become hot spots in the field of antiviral research. NeST was shown to enhance IFN-␥ production, controlling the susceptibility of mice to persistent Theiler's virus infection as well as resistance to Salmonella enterica serovar Typhimurium infection . . Both CMPK2 and NRAV were identified as negative regulators of IFN immune reactions. CMPK2, induced by IFN-␣ or HCV infection, suppresses various ISGs, the knockdown of which dramatically blocks HCV replication . . NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . .",". NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . . Numerous lncRNAs, including lnc-ISG15 and ISR2, respond to IFNs such as ISGs, although their actual function requires further investigation . . Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection.","Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection. NEAT1 has been reported to interact with Drosophila DBHS RNA-binding proteins e.g., SFPQ, NONO-p54nrb, and PSPC1 , recruiting them to paraspeckles, a nuclear substructure found in all cultured and primary cells except embryonic stem cells . . The versatile function of NEAT1 is rapidly progressing in multiple areas of biology. NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . .","NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . . NEAT1 also participates in neurodegenerative diseases such as Huntington's disease . and seems to potentially contribute to the elevated production of a number of cytokines and chemokines in patients with systemic lupus erythematosus SLE . . Furthermore, poly I·C can activate NEAT1 transcription through the TLR3 pathway, whereas NEAT1 positively regulates IL-8 transcription and potentially affects the expression of multiple ISGs after poly I·C stimulation . . In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . .","In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . . However, the role of NEAT1 in hantaviral infection remains unclear. In this report, NEAT1 has been identified as an important regulator of the host innate immune system against HTNV infection. Elevated NEAT1 promotes IFN secretion, most likely by enhancing RIG-I and DDX60 expression. DDX60, a DEXD/H box RNA helicase similar to Saccharomyces cerevisiae Ski2, is induced after viral infection . . DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response.",". DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response. However, the antiviral effects of DDX60 seem to vary among viruses . . We found that NEAT1-regulated DDX60 was involved in IFN production in response to HTNV infection. In HTNV-infected cells, double-stranded RNA dsRNA could not be detected, and it is unclear how host PRRs, especially RIG-I, recognize HTNV invasion . . Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation.",". Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation. Of note, we applied multiple cell lines to explore the role of NEAT1 during HTNV infection. HTNV primarily targets vascular endothelial cells in vivo and contributes to the increased vascular permeability and coagulation disorders in HFRS; hence, HUVECs are the most common in vitro cell model to study host innate immunity against HTNV infection or viral pathogenesis . . EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies .",". EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies . . A549 cells were once used to isolate HTNV, and they were confirmed to be a mature model of infection . . . . . Additionally, Huh 7.0 and Huh 7.5 RIG-I Ϫ cells used in our study have been reported to be infected by HTNV by Lee et al. . and can be used as a cell model to study immune responses against HTNV replication . . Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV.",". Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV. Using qRT-PCR, Western blotting, and immunofluorescence assays, we have also shown that both HEK293 and HeLa cells can be infected by HTNV. To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production.","To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production. Alterations in the relative fluorescence intensity of NP after silencing or overexpressing NEAT1-2 did not seem to be as remarkable as qRT-PCR or Western blot analysis results. The NP spotted and exhibited in the ICW results forms obvious stains that mimic PFU. However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays.","However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays. The RNAi studies in vivo are encouraging Fig. 8 , but the NC used by our group was not mutated si-NEAT1-2 i.e., same sense strand, but with a point mutation in the targeting strand . The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling.","The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling. After observing that NEAT1 can regulate IFN expression by HTNV infection, we were interested in the function of NEAT1. We noticed that silencing NEAT1-2 or ectopically expressing NEAT1-2 could not inhibit or enhance IFN expression without HTNV infection Fig. 3F , which indicated that NEAT1-2 could not directly affect IFN- expression. This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression.","This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression. Recent findings have shown that RIG-I signaling is essential for an efficient polyfunctional T cell response during IAV infection . . Indeed, we found that the function of T cells was suppressed after NEAT1-2 depletion in our animal experiments Fig. 8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection.","8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection. HTNV infection induced NEAT1 expression through the RIG-I-IRF7 pathway, while NEAT1 displayed positive feedback for RIG-I signaling. NEAT1 relocated SFPQ from the potential promoter region of several antiviral genes to the paraspeckles, removing the transcriptional inhibitory effects of SFPQ. This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 .","This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 . nontarget control i.e., negative control NC or targeted RIG-I and DDX60 were designed by Gene-Pharma as follows: NC, 5=- UUCUUCGAACGUGUCACGUTT-3=; si-RIG-I-1, 5=-GCCCAUUUAAACCAAGAAATT-3=, si-RIG-I-2, 5=-GGUGGAGGAUAUUUGAACUTT-3=, and si-RIG-I-3, 5=-CCCAACGAUAUCAUUUCUTT-3=; si-DDX60-1, 5=-GUCCAGGUGUCAGUUUGAUTT-3=, si-DDX60-2, 5=-CCGAAGUGAAGAAGGUAAATT-3=, and si-DDX60-3, 5=-GAUGGAUGCUAGGAAAUAUTT-3=. The pCMV-NEAT1-1 and pCMV-NEAT1-2 plasmids, which transcribe NEAT1-1 and NEAT1-2, respectively, were provided by Nakagawa Shinichi . . The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . .",". The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . . Abs against RIG-I, IRF3, IRF7, p65, and STAT1, as well as the neutralizing antibodies against IFN-␣ and IFN-, were purchased from Abcam Cambridge, MA, USA . Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China .","Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China . The Abs targeting CD11b, F4/80, CD3, CD8, IFN-␥, inducible nitric oxide synthase iNOS , and CD206 for flow cytometry were purchased from BD Biosciences San Jose, CA, USA . IFN-␣, -, and -␥, TNF-␣, and IL-1 were from PeperoTech Rocky Hill, NJ . ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis.","ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis. Total cellular RNAs were extracted with RNAiso TaKaRa, Dalian, China , the concentration of which was measured using a NanoDrop 1000 spectrophotometer. Reverse transcription RT was then performed with PrimeScript RT master mix TaKaRa according to the instructions provided by the manufacturer. Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed.","Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed. The qRT-PCR primer sequences for NEAT1, NEAT1-2, IFN-, HTNV S segment, RIG-I, DDX60, -actin, and GAPDH were obtained from previous reports . . The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing.",". The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing. HUVECs with a confluence of 80% in 6 wells were mock infected or infected with live or 60 Co-inactivated HTNV at an MOI of 1. RNAs were extracted as previously described at 24 hpi, and the quality was analyzed using FastQC software by the Beijing Genomics Institute BGI, Shenzhen, China . Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded.","Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded. Tag counts were normalized to TPM transcripts per million by dividing the raw tag count by the total number of tags from each library and multiplying by 1 million. To avoid the possible noise signal from high-throughput sequencing, the genes with average TPM of less than 1 in these three states were excluded. In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01.","In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01. Genes were selected as differentially expressed using a P value threshold of 0.01. FISH and immunofluorescence assays IFA . Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature.","Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature. Prehybridization was performed with lncRNA FISH probe mix at 37°C for 30 min, and then hybridization was performed by adding NEAT1-2 FISH probe mix and incubating the mixture at 37°C overnight. After washing with 4ϫ, 2ϫ, and 1ϫ SSC, the cell nuclei were stained with DAPI 4=,6-diamidino-2-phenylindole . Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently.","Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently. The cells were fixed with 4% PFA for 10 min and permeabilized with 0.1% Triton X-100 for 15 min. Primary Abs were added and incubated at 37°C for 2 h. After five washes with DPBS, secondary Cy3-or fluorescein isothiocyanate FITC -conjugated goat anti-rabbit or goat anti-mouse IgG Sangon, Shanghai, China was added and incubated at 37°C for 2 h. Cell nuclei were stained with DAPI. Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue .","Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue . The samples were then boiled at 95°C for 10 min. The lysates were resolved by 10%, 12%, or 15% SDS-PAGE and transferred to polyvinylidene fluoride PVDF membranes Millipore . The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA .","The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA . ICW assay. The In-Cell Western ICW assay was performed using an Odyssey imaging system Li-Cor according to the manufacturer's instructions. HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1.","HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1. At 48 hpi, HUVECs were washed twice with ice-cold DPBS, fixed with 4% PFA for 10 min, and permeabilized with 1.0% Triton X-100 for 15 min. Cells were added with Li-Cor Odyssey blocking solution at room temperature for 30 min and incubated at 4°C overnight with mouse IgG MAb 1A8 against HTNV NP together with rabbit IgG antibody against -actin, both of which were diluted in PBS containing 3% bovine serum albumin BSA; HyClone . Subsequently, the cells were washed and stained with goat anti-mouse IgG IRDye 800 antibody 1:5,000; Li-Cor and goat anti-rabbit"],"string":"[\n \"Hantavirus infection, which causes zoonotic diseases with a high mortality rate in humans, has long been a global public health concern. Over the past decades, accumulating evidence suggests that long noncoding RNAs lncRNAs play key regulatory roles in innate immunity. However, the involvement of host lncRNAs in hantaviral control remains uncharacterized. In this study, we identified the lncRNA NEAT1 as a vital antiviral modulator. NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection.\",\n \"NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection. Further investigation suggested that NEAT1 served as positive feedback for RIG-I signaling. HTNV infection activated NEAT1 transcription through the RIG-I–IRF7 pathway, whereas NEAT1 removed the transcriptional inhibitory effects of the splicing factor proline- and glutamine-rich protein SFPQ by relocating SFPQ to paraspeckles, thus promoting the expression of RIG-I and DDX60. RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections.\",\n \"RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections. IMPORTANCE Hantaviruses have attracted worldwide attention as archetypal emerging pathogens. Recently, increasing evidence has highlighted long noncoding RNAs lncRNAs as key regulators of innate immunity; however, their roles in hantavirus infection remain unknown. In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling.\",\n \"In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling. This lncRNA was induced by HTNV through the RIG-I–IRF7 pathway in a time- and dose-dependent manner and promoted HTNV-induced IFN production by facilitating RIG-I and DDX60 expression. Intriguingly, NEAT1 relocated SFPQ and formed paraspeckles after HTNV infection, which might reverse inhibitive effects of SFPQ on the transcription of RIG-I and DDX60. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections.\",\n \"To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections. Text: glycoprotein GP , and viral RNA-dependent polymerase protein RdRp , respectively. Humans become infected by inhaling contaminated aerosols or by coming into contact with rodent excreta, and they develop two severe acute diseases, namely, hemorrhagic fever with renal syndrome HFRS and hantavirus pulmonary syndrome HPS . . Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . .\",\n \". Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . . Chinese HFRS cases, mainly caused by Hantaan virus HTNV infection, account for approximately 90% of all global cases, with a mortality rate ranging from 0.1 to 15% . . Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions.\",\n \"Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions. Cellular pathogen recognition receptors PRRs , including Toll-like receptors TLRs and RIG-I like receptors RLRs , can detect distinct pathogen-associated molecular patterns PAMPs and trigger the expression of IFNs and cytokines. RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression.\",\n \"RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression. lncRNA nuclear paraspeckle assembly transcript 1 NEAT1 is an essential architectural constituent of paraspeckles in the mammalian nucleus, interacting with Drosophila DBHS RNA-binding proteins such as the splicing factor proline-and glutamine-rich protein SFPQ and the non-POU domain-containing, octamer-binding protein NONO/p54 . . To date, two isoform transcripts of the NEAT1 gene have been identified, namely, the 3.7-kb NEAT1-1 MEN and the 23-kb NEAT1-2 MEN Fig. 1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . .\",\n \"1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . . , promoting cancer formation in mice by dampening oncogene-dependent activation of p53 . . Nevertheless, studies assessing the function of NEAT1 in viral infections are scarce. Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication.\",\n \"Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication. Further investigation showed that NEAT1 promoted RIG-I and DDX60 expression by relocating SFPQ and removing the transcriptional inhibitory effects of SFPQ, which are critical for IFN responses against HTNV infection. We also found that RIG-I signaling, rather than TLR3 and TLR4, accounted for the elevation of HTNV-induced NEAT1. Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection.\",\n \"Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection. As shown in Fig. 1B , the NEAT1 level in the HTNV group was higher than that in the mock group P ϭ 6.86 ϫ 10 Ϫ13 , false discovery rate FDR ϭ 9.75 ϫ 10 Ϫ12 or the 60 Co-inactivated HTNV group P ϭ 1.75 ϫ 10 Ϫ14 , FDR ϭ 3.10 ϫ 10 Ϫ13 ; however, the difference between the 60 Co-inactivated HTNV group and the mock group was not significant P ϭ 0.21034, FDR ϭ 0.58211 . To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig.\",\n \"To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig. 1A , were applied to quantify NEAT1 RNA isoforms by quantitative real-time PCR qRT-PCR . It has been reported that NEAT1-2 rather than NEAT1-1 plays a key regulatory role in paraspeckle formation . , and we also found that elevated NEAT1 levels depend on live HTNV infection rather than 60 Co-inactivated HTNV stimulation Fig. 1C . Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D .\",\n \"Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D . To further investigate whether NEAT1 expression was altered in other cell lines, HEK293, HeLa, and A549 cells were used. All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G .\",\n \"All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G . Similar to the data obtained from HUVECs, NEAT1 was indeed upregulated by HTNV at a multiplicity of infection MOI of 1 beginning at 24 hpi in HUVECs and A549, HEK293, and HeLa cells, and the increasing tendency occurred in a time-dependent manner Fig. 1H . Of note, the NEAT1 elevation at 2 hpi might have been unrelated to the virus but resulted in cellular stress responses. Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I .\",\n \"Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I . NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. The abovedescribed data showed that HTNV infection increased NEAT1, and we wondered how NEAT1 could reciprocally influence HTNV replication. The small interfering RNA siRNA transfection efficiency in HUVECs was confirmed by flow cytometry, and NEAT1 expression was significantly decreased, as assessed by qRT-PCR after RNA interference RNAi Fig. 2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2.\",\n \"2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2. Compared with the cells transfected with control siRNA negative control NC , HUVECs with si-NEAT1 could dramatically promote HTNV NP production, and NP expression seemed to be related to the amount of applied si-NEAT1 Fig. 2B . Intriguingly, depletion of NEAT1-2 alone could mimic the antiviral effects of simultaneous NEAT1-1 and NEAT1-2 silencing Fig. 2C , indicating that NEAT1-2 was critical for the antiviral responses. Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing.\",\n \"Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing. On the other hand, plasmids, of which pCMV-NEAT1-1 is transcribed into the 3.7-kb NEAT1-1 MEN and pCMV-NEAT1-2 is transcribed into the 2-to 3-kb NEAT1-2 MEN , were applied to directly investigate the role of NEAT1 in HTNV infection Fig. 2F . Surprisingly, we found NEAT1-1 overexpression restricted NEAT1-2 transcription Fig. 2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G .\",\n \"2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G . Furthermore, overexpression of NEAT1-2 instead of NEAT1-1 could efficiently suppress HTNV replication Fig. 2H . NEAT1-1 upregulation even aggravated HTNV infection Fig. 2H , which may be the result of downregulation of NEAT1-2. Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig.\",\n \"Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig. 2J were limited in HUVECs in which NEAT1-2 was ectopically expressed in comparison to those transfected with control vector or pCMV-NEAT1-1. These data further showed that NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. Alteration of NEAT1-2 affects HTNV-induced IFN expression in HUVECs. IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . .\",\n \"IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . . It has been reported that the GnT of hantaviruses suppressed IFN- expression of host cells at an early stage of infection . . Here, we also found that HUVECs could not efficiently produce IFN- until 12 hpi at an MOI of 0.1 or until 24 hpi at an MOI of 1 Fig. 3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig.\",\n \"3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig. 3B , suggesting that NEAT1-2 expression increased just before IFN production. We found that expression of endogenous IFN- mRNA was much lower in cells transfected with si-NEAT1-2 at MOIs of both 0.1 Fig. 3C and 1 Fig. 3D than in those transfected with control siRNA NC . In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E .\",\n \"In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E . More importantly, HUVECs transfected with pCMV-NEAT1-2 conspicuously increased IFN- gene expression compared with those cells with vector plasmids at 12 hpi MOI ϭ 1 , demonstrating that NEAT1-2 overexpression accelerated robust IFN responses in host cells against HTNV infection. With a dual luciferase reporter system Twenty-four hours after transfection, the cells expressing FAM were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1.\",\n \"Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the NC group . NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g .\",\n \"NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 0.1 for 48 h. The expression of HTNV NP was measured by Western blotting. C HUVECs were treated as described for panel A, right, but at an MOI of 0.1. In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.\",\n \"In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NC group . D HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group .\",\n \"The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . E HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g .\",\n \"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g . Twenty-four hours after transfection, the cells expressing green fluorescent protein GFP were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with control plasmids vector , pCMV-NEAT1-1, or pCMV-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig.\",\n \"Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig. 3F . These results showed that NEAT1-2 regulated HTNV-induced IFN- expression. To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig.\",\n \"To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig. 3G . In addition, in cells with high NEAT1-2 expression, treatment with neutralizing antibodies NAbs of IFN-␣ and IFN- could counteract the antiviral effects of NEAT1-2 MOI ϭ 1 , and the compensatory effects were dependent on the magnitude of the NAbs. Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production.\",\n \"Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production. PRRs maintain a vital role in the promotion of IFN responses, and we conjectured that NEAT1 might amplify IFN responses by modulating these molecules. TLR3, TLR4, and RIG-I have been shown to recognize HTNV infection . . DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate .\",\n \". DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate . , Here, we found that multiple Toll-like receptors like TLR1, TLR2, TLR3, and TLR4, as well as MDA5, were increased after HTNV infection, but none of them were influenced by silencing NEAT1-2 Fig. 4A . The upregulated RIG-I and DDX60 were blocked in the cells with low NEAT1-2 expression after HTNV infection Fig. 4A . HUVECs with declining NEAT1-2 expression showed gradually decreasing expression of RIG-I and DDX60 Fig. 4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C .\",\n \"4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C . These data indicated that NEAT1-2 could positively modulate RIG-I and DDX60 expression, while the role of RIG-I and DDX60 upon HTNV infection is obscure. We then found that RIG-I and DDX60 colocalized after HTNV infection Fig. 4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown .\",\n \"4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown . Simultaneously knocking down RIG-I and DDX60 significantly promoted HTNV NP expression Fig. 4E , and knockdown of both of them could greatly affect IFN- expression Fig. 4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig.\",\n \"4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig. 4H , indicating that efficient anti-HTNV responses might depend on the interactive effects of DDX60 and RIG-I. More importantly, RIG-I or/and DDX60 overexpression enhanced HTNV-induced IFN- expression, and they had synergistic effects on IFN- production Fig. 4I and J . Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60.\",\n \"Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60. NEAT1 was found to interact with SFPQ by RNA immunoprecipitation RIP after HTNV infection Fig. 5A , indicating that modulatory effects of NEAT1 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively .\",\n \"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1 for 48 h. The expression of HTNV NP was measured by Western blotting. H HUVECs were treated as described for panel F, right. In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.\",\n \"In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . H HUVECs were treated as described for panel F, right. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group .\",\n \"The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . I HUVECs were treated as described for panel F, right. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ.\",\n \"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ. Interestingly, the protein level of SFPQ, as well as another paraspeckle-forming constituent, NONO, remained unchanged after HTNV infection Fig. 5B or after NEAT1 overexpression and knockdown Fig. 5C . However, SFPQ became centralized rather than diffuse in the nucleus after HTNV infection Fig. 5D . The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig.\",\n \"The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig. 5F and G , which might have been related to the increase in RIG-I Fig. 5H and DDX60 Fig. 5I . SFPQ has been suggested to bind to the promoter region of RIG-I and DDX60 ., thus preventing the expression of RIG-I and DDX60. Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription.\",\n \"Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription. Interestingly, overexpression of the S or M segment of HTNV in HEK293 cells failed to induce NEAT1 expression, suggesting that NEAT1 transcription was closely related to live viral replication Fig. 6A . Of note, the upregulation of NEAT1 by HTNV could not be reversed by applying IFN-I neutralizing antibodies Fig. 6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig.\",\n \"6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig. 6C, D, and E or cytokines Fig. 6F and G . We conjectured that NEAT1 expression was related to the activation of PRRs. By knocking down several PRRs, we found that the RIG-I and TLR4 pathways played important roles in HTNV-induced NEAT1 upregulation Fig. 7A . Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C .\",\n \"Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C . Moreover, using STAT1 as a positive control, we found that the transcription factor IRF7, rather than IRF3 and p65, translocated into the nucleus in HTNV-infected HUVECs at 2 dpi Fig. 7D . Furthermore, IRF7 knockdown blocked HTNV-induced NEAT1 upregulation Fig. 7E . Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice.\",\n \"Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice. Although cell-based experiments revealed that NEAT1-2 is a crucial regulator of innate antihantaviral responses, its function in vivo has remained unclear. To address this question, we intravenously injected siRNAs targeting mouse NEAT1-2 at 1 day before HTNV infection. NEAT1-2 expression levels in the liver, kidney, and spleen were reduced at 2 dpi Fig. 8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions.\",\n \"8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions. Body weight loss in NEAT1-2-depleted mice was observed from 2 dpi to 5 dpi, and the IFN production in serum was remarkably decreased in the NEAT1-2 silenced group than those in the NC group at 3 dpi Fig. 8B . As expected, NEAT1-2 knockdown mice showed considerably higher HTNV NP levels in the liver, spleen, and kidney at 3 dpi Fig. 8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D .\",\n \"8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D . In addition, reduced inflammatory cell filtration but increased tissue injury was found in NEAT1-2 knockdown mice during the early stage of infection Fig. 8E . Infiltration of macrophages in the spleen was attenuated Fig. 8F , and the activation of macrophages was also suppressed by flow cytometry; data not shown . Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G .\",\n \"Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G . Nevertheless, NEAT1-2 silencing had no effect on the production of neutralizing antibodies at 7 dpi data not shown . The above-described findings indicated that NEAT1-2 depletion might influence multiple aspects of the innate immune response in HTNV-infected mice. Innate immunity is a phylogenetically ancient and conserved system that counteracts invading microbes, the regulatory mechanism of which is sophisticated and complex. Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . .\",\n \"Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . . In this report, we first demonstrated that NEAT1 was induced by HTNV through the RIG-I-IRF7 pathway and served as positive feedback for RIG-I signaling. Using DGE analysis, we observed upregulated NEAT1 and confirmed its alteration in To further determine the function of NEAT1 after HTNV infection in vivo, mice were injected intravenously with si-NEAT1-2 1 g/g or nontarget control siRNA NC 1 g/g ; 1 day later, they were infected with HTNV 100 LD 50 by intramuscular injection. A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group .\",\n \"A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group . B The effects of NEAT1 on HTNV virulence in mice were determined by body weight loss from 0 to 10 dpi left panel, n ϭ 10 in each group . The IFN- in sera of different groups was measured by ELISA at 3dpi right panel, n ϭ 8 in each group . Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi.\",\n \"Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi. Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . D NEAT1 effects on HTNV infection kinetics at 3 dpi were determined by testing the HTNV titers in livers, spleens, and kidneys. Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group .\",\n \"Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group . After HTNV infection, livers in the NC group showed inflammatory cell infiltration in certain regions, while those in the si-NEAT1-2 group showed slight acute viral hepatitis. Spleens in the NC group showed lymph node hyperplasia, while those in the si-NEAT1-2 group were severely congestive. Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented.\",\n \"Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented. G CD3 ϩ CD8 ϩ IFN-␥ ϩ T cells were analyzed by flow cytometry at 3 dpi, and the results obtained for three mice in each group are presented. different cell lines. To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression.\",\n \"To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression. By virtue of RNAi, the RIG-I-IRF7 pathway was confirmed to be necessary for HTNV-triggered NEAT1 elevation. Recently, large-scale transcriptomic studies identified numerous noncoding transcripts in the mammalian genome, which were speculated to influence diverse biological processes. Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression .\",\n \"Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression . . TLR2 activation or tumor necrosis factor alpha TNF-␣ stimulation induces transcription of the lncRNA THRIL, the downregulation of which impairs TNF-␣ and IL-6 secretion . . TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . .\",\n \". TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . . The lncRNA Lethe, triggered by TNF-␣ and IL-1, acts as a negative feedback regulator of NF-B signaling . . The roles of lncRNAs in host-virus interactions have been progressively unveiled. Various viruses, such as influenza virus IAV , coronavirus, enterovirus, human immunodeficiency virus HIV , hepatitis B virus HBV , hepatitis C virus HCV , Japanese encephalitis virus JEV , and rabies virus, have been reported to activate the transcription of different lncRNAs in host cells 11, . . . .\",\n \". . . Importantly, multiple lncRNAs have been shown to affect the IFN response in recent years and have gradually become hot spots in the field of antiviral research. NeST was shown to enhance IFN-␥ production, controlling the susceptibility of mice to persistent Theiler's virus infection as well as resistance to Salmonella enterica serovar Typhimurium infection . . Both CMPK2 and NRAV were identified as negative regulators of IFN immune reactions. CMPK2, induced by IFN-␣ or HCV infection, suppresses various ISGs, the knockdown of which dramatically blocks HCV replication . . NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . .\",\n \". NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . . Numerous lncRNAs, including lnc-ISG15 and ISR2, respond to IFNs such as ISGs, although their actual function requires further investigation . . Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection.\",\n \"Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection. NEAT1 has been reported to interact with Drosophila DBHS RNA-binding proteins e.g., SFPQ, NONO-p54nrb, and PSPC1 , recruiting them to paraspeckles, a nuclear substructure found in all cultured and primary cells except embryonic stem cells . . The versatile function of NEAT1 is rapidly progressing in multiple areas of biology. NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . .\",\n \"NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . . NEAT1 also participates in neurodegenerative diseases such as Huntington's disease . and seems to potentially contribute to the elevated production of a number of cytokines and chemokines in patients with systemic lupus erythematosus SLE . . Furthermore, poly I·C can activate NEAT1 transcription through the TLR3 pathway, whereas NEAT1 positively regulates IL-8 transcription and potentially affects the expression of multiple ISGs after poly I·C stimulation . . In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . .\",\n \"In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . . However, the role of NEAT1 in hantaviral infection remains unclear. In this report, NEAT1 has been identified as an important regulator of the host innate immune system against HTNV infection. Elevated NEAT1 promotes IFN secretion, most likely by enhancing RIG-I and DDX60 expression. DDX60, a DEXD/H box RNA helicase similar to Saccharomyces cerevisiae Ski2, is induced after viral infection . . DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response.\",\n \". DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response. However, the antiviral effects of DDX60 seem to vary among viruses . . We found that NEAT1-regulated DDX60 was involved in IFN production in response to HTNV infection. In HTNV-infected cells, double-stranded RNA dsRNA could not be detected, and it is unclear how host PRRs, especially RIG-I, recognize HTNV invasion . . Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation.\",\n \". Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation. Of note, we applied multiple cell lines to explore the role of NEAT1 during HTNV infection. HTNV primarily targets vascular endothelial cells in vivo and contributes to the increased vascular permeability and coagulation disorders in HFRS; hence, HUVECs are the most common in vitro cell model to study host innate immunity against HTNV infection or viral pathogenesis . . EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies .\",\n \". EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies . . A549 cells were once used to isolate HTNV, and they were confirmed to be a mature model of infection . . . . . Additionally, Huh 7.0 and Huh 7.5 RIG-I Ϫ cells used in our study have been reported to be infected by HTNV by Lee et al. . and can be used as a cell model to study immune responses against HTNV replication . . Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV.\",\n \". Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV. Using qRT-PCR, Western blotting, and immunofluorescence assays, we have also shown that both HEK293 and HeLa cells can be infected by HTNV. To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production.\",\n \"To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production. Alterations in the relative fluorescence intensity of NP after silencing or overexpressing NEAT1-2 did not seem to be as remarkable as qRT-PCR or Western blot analysis results. The NP spotted and exhibited in the ICW results forms obvious stains that mimic PFU. However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays.\",\n \"However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays. The RNAi studies in vivo are encouraging Fig. 8 , but the NC used by our group was not mutated si-NEAT1-2 i.e., same sense strand, but with a point mutation in the targeting strand . The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling.\",\n \"The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling. After observing that NEAT1 can regulate IFN expression by HTNV infection, we were interested in the function of NEAT1. We noticed that silencing NEAT1-2 or ectopically expressing NEAT1-2 could not inhibit or enhance IFN expression without HTNV infection Fig. 3F , which indicated that NEAT1-2 could not directly affect IFN- expression. This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression.\",\n \"This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression. Recent findings have shown that RIG-I signaling is essential for an efficient polyfunctional T cell response during IAV infection . . Indeed, we found that the function of T cells was suppressed after NEAT1-2 depletion in our animal experiments Fig. 8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection.\",\n \"8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection. HTNV infection induced NEAT1 expression through the RIG-I-IRF7 pathway, while NEAT1 displayed positive feedback for RIG-I signaling. NEAT1 relocated SFPQ from the potential promoter region of several antiviral genes to the paraspeckles, removing the transcriptional inhibitory effects of SFPQ. This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 .\",\n \"This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 . nontarget control i.e., negative control NC or targeted RIG-I and DDX60 were designed by Gene-Pharma as follows: NC, 5=- UUCUUCGAACGUGUCACGUTT-3=; si-RIG-I-1, 5=-GCCCAUUUAAACCAAGAAATT-3=, si-RIG-I-2, 5=-GGUGGAGGAUAUUUGAACUTT-3=, and si-RIG-I-3, 5=-CCCAACGAUAUCAUUUCUTT-3=; si-DDX60-1, 5=-GUCCAGGUGUCAGUUUGAUTT-3=, si-DDX60-2, 5=-CCGAAGUGAAGAAGGUAAATT-3=, and si-DDX60-3, 5=-GAUGGAUGCUAGGAAAUAUTT-3=. The pCMV-NEAT1-1 and pCMV-NEAT1-2 plasmids, which transcribe NEAT1-1 and NEAT1-2, respectively, were provided by Nakagawa Shinichi . . The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . .\",\n \". The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . . Abs against RIG-I, IRF3, IRF7, p65, and STAT1, as well as the neutralizing antibodies against IFN-␣ and IFN-, were purchased from Abcam Cambridge, MA, USA . Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China .\",\n \"Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China . The Abs targeting CD11b, F4/80, CD3, CD8, IFN-␥, inducible nitric oxide synthase iNOS , and CD206 for flow cytometry were purchased from BD Biosciences San Jose, CA, USA . IFN-␣, -, and -␥, TNF-␣, and IL-1 were from PeperoTech Rocky Hill, NJ . ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis.\",\n \"ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis. Total cellular RNAs were extracted with RNAiso TaKaRa, Dalian, China , the concentration of which was measured using a NanoDrop 1000 spectrophotometer. Reverse transcription RT was then performed with PrimeScript RT master mix TaKaRa according to the instructions provided by the manufacturer. Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed.\",\n \"Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed. The qRT-PCR primer sequences for NEAT1, NEAT1-2, IFN-, HTNV S segment, RIG-I, DDX60, -actin, and GAPDH were obtained from previous reports . . The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing.\",\n \". The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing. HUVECs with a confluence of 80% in 6 wells were mock infected or infected with live or 60 Co-inactivated HTNV at an MOI of 1. RNAs were extracted as previously described at 24 hpi, and the quality was analyzed using FastQC software by the Beijing Genomics Institute BGI, Shenzhen, China . Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded.\",\n \"Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded. Tag counts were normalized to TPM transcripts per million by dividing the raw tag count by the total number of tags from each library and multiplying by 1 million. To avoid the possible noise signal from high-throughput sequencing, the genes with average TPM of less than 1 in these three states were excluded. In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01.\",\n \"In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01. Genes were selected as differentially expressed using a P value threshold of 0.01. FISH and immunofluorescence assays IFA . Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature.\",\n \"Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature. Prehybridization was performed with lncRNA FISH probe mix at 37°C for 30 min, and then hybridization was performed by adding NEAT1-2 FISH probe mix and incubating the mixture at 37°C overnight. After washing with 4ϫ, 2ϫ, and 1ϫ SSC, the cell nuclei were stained with DAPI 4=,6-diamidino-2-phenylindole . Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently.\",\n \"Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently. The cells were fixed with 4% PFA for 10 min and permeabilized with 0.1% Triton X-100 for 15 min. Primary Abs were added and incubated at 37°C for 2 h. After five washes with DPBS, secondary Cy3-or fluorescein isothiocyanate FITC -conjugated goat anti-rabbit or goat anti-mouse IgG Sangon, Shanghai, China was added and incubated at 37°C for 2 h. Cell nuclei were stained with DAPI. Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue .\",\n \"Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue . The samples were then boiled at 95°C for 10 min. The lysates were resolved by 10%, 12%, or 15% SDS-PAGE and transferred to polyvinylidene fluoride PVDF membranes Millipore . The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA .\",\n \"The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA . ICW assay. The In-Cell Western ICW assay was performed using an Odyssey imaging system Li-Cor according to the manufacturer's instructions. HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1.\",\n \"HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1. At 48 hpi, HUVECs were washed twice with ice-cold DPBS, fixed with 4% PFA for 10 min, and permeabilized with 1.0% Triton X-100 for 15 min. Cells were added with Li-Cor Odyssey blocking solution at room temperature for 30 min and incubated at 4°C overnight with mouse IgG MAb 1A8 against HTNV NP together with rabbit IgG antibody against -actin, both of which were diluted in PBS containing 3% bovine serum albumin BSA; HyClone . Subsequently, the cells were washed and stained with goat anti-mouse IgG IRDye 800 antibody 1:5,000; Li-Cor and goat anti-rabbit\"\n]"},"document_id":{"kind":"number","value":2652,"string":"2,652"},"id":{"kind":"number","value":5313,"string":"5,313"}}},{"rowIdx":674,"cells":{"question":{"kind":"string","value":"What serious question was raised?"},"answer":{"kind":"string","value":"as to whether or not our seasonal or pandemic flu might have another reservoir host."},"context_chunks":{"kind":"list like","value":["nan Text: pecially with next-generation sequencing NGS for new virus genome discovery, e.g., Ruben Donis et al. sequenced a bat-derived influenza virus genome by using NGS in 2012, raising a serious question as to whether or not our seasonal or pandemic flu might have another reservoir host. Chen and colleagues confirmed the SFTSV independently by using NGS. Indeed, metagenomics analysis has yielded a great deal of new viruses, especially from the environment. Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal .","Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal . In this issue, six groups have been invited to present their recent findings on the emerging viruses, in addition to a previous report on H7N9 . Shi reviewed recent discoveries of new viruses or virus genomes from bat. Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 .","Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 . Recent MERS-CoV infection is another example for severe disease caused by used-to-be-less pathogenic coronaviruses. Shi and colleagues by using NGS have discovered many unknown animal viruses from bat, especially some important paramyxoviruses and reoviruses. Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city .","Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city . The potential roles of these viruses in bats for interspecies transmission are yet to be elucidated. Tan and colleagues specifically focused on the newly-emerged MERS-CoV. The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries.","The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries. In the initial diagnosis, the pan-coronavirus real-time reverse transcription polymerase chain reaction RT-PCR assay played a very important role for the identification of the causative agents. By using this method, scientists detected an expected-size PCR fragment for the corresponding conserved region of ORF1b of the replicase gene of a coronavirus. This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently .","This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently . Enterovirus has been known as serious human pathogens for a long time but their significance to the public health has been emphasized by the emergence of enterovirus 71 in 1998 as a serious pathogenic agents for children in Taiwan and re-emerged in mainland China in 2008 . In this issue, Duan and colleagues summarized the findings of new enteroviruses by using NGS. Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses.","Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses. The review also provided a detailed description of the NGS and related molecular methods for the virus discovery followed by a list of new enteroviruses found in human feces. These include viruses in the family of Piconaviridae, Parvoviridae, Circoviridae, Astroviridae and Polyomaviridae. Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future.","Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future. The disease caused by SFTSV, with a CFR of 12%, had been in China for a couple of years before the causative agent was finally identified. There are still a lot of questions remained unknown for this new virus and vigorous studies are in great need. The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts.","The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts. Therefore the effective control measures are still under evaluation. Vaccines protecting the SFTSV infection are under its way in Chinese Center for Disease Control and Prevention. Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered.","Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered. The new flavivirus, duck egg-drop syndrome virus DEDSV , is a good example. Su and colleagues reviewed the characterization of the DEDSV and its disease form in this issue. The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese .","The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese . Yet the transmission vector, though mosquitoes are suspected, has not been identified. Due to the public health concerns of its related viruses, potential human infection of DEDSV should be evaluated. Research on insect viruses is reviving in recent years. In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses.","In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses. Studies on these viruses can provide useful knowledge for our understanding about animal or human infecting viruses. More importantly, modification and application of insect-infecting viruses can be used as effective biologicals for the control of insect pest. The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing.","The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing. Human beings encounter more ecology-climate-changing problems, including the zoonotic pathogens. We have to face some unknown pathogenic agents passively. To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown.","To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown. This is especially true for infectious agents."],"string":"[\n \"nan Text: pecially with next-generation sequencing NGS for new virus genome discovery, e.g., Ruben Donis et al. sequenced a bat-derived influenza virus genome by using NGS in 2012, raising a serious question as to whether or not our seasonal or pandemic flu might have another reservoir host. Chen and colleagues confirmed the SFTSV independently by using NGS. Indeed, metagenomics analysis has yielded a great deal of new viruses, especially from the environment. Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal .\",\n \"Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal . In this issue, six groups have been invited to present their recent findings on the emerging viruses, in addition to a previous report on H7N9 . Shi reviewed recent discoveries of new viruses or virus genomes from bat. Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 .\",\n \"Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 . Recent MERS-CoV infection is another example for severe disease caused by used-to-be-less pathogenic coronaviruses. Shi and colleagues by using NGS have discovered many unknown animal viruses from bat, especially some important paramyxoviruses and reoviruses. Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city .\",\n \"Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city . The potential roles of these viruses in bats for interspecies transmission are yet to be elucidated. Tan and colleagues specifically focused on the newly-emerged MERS-CoV. The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries.\",\n \"The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries. In the initial diagnosis, the pan-coronavirus real-time reverse transcription polymerase chain reaction RT-PCR assay played a very important role for the identification of the causative agents. By using this method, scientists detected an expected-size PCR fragment for the corresponding conserved region of ORF1b of the replicase gene of a coronavirus. This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently .\",\n \"This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently . Enterovirus has been known as serious human pathogens for a long time but their significance to the public health has been emphasized by the emergence of enterovirus 71 in 1998 as a serious pathogenic agents for children in Taiwan and re-emerged in mainland China in 2008 . In this issue, Duan and colleagues summarized the findings of new enteroviruses by using NGS. Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses.\",\n \"Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses. The review also provided a detailed description of the NGS and related molecular methods for the virus discovery followed by a list of new enteroviruses found in human feces. These include viruses in the family of Piconaviridae, Parvoviridae, Circoviridae, Astroviridae and Polyomaviridae. Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future.\",\n \"Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future. The disease caused by SFTSV, with a CFR of 12%, had been in China for a couple of years before the causative agent was finally identified. There are still a lot of questions remained unknown for this new virus and vigorous studies are in great need. The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts.\",\n \"The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts. Therefore the effective control measures are still under evaluation. Vaccines protecting the SFTSV infection are under its way in Chinese Center for Disease Control and Prevention. Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered.\",\n \"Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered. The new flavivirus, duck egg-drop syndrome virus DEDSV , is a good example. Su and colleagues reviewed the characterization of the DEDSV and its disease form in this issue. The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese .\",\n \"The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese . Yet the transmission vector, though mosquitoes are suspected, has not been identified. Due to the public health concerns of its related viruses, potential human infection of DEDSV should be evaluated. Research on insect viruses is reviving in recent years. In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses.\",\n \"In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses. Studies on these viruses can provide useful knowledge for our understanding about animal or human infecting viruses. More importantly, modification and application of insect-infecting viruses can be used as effective biologicals for the control of insect pest. The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing.\",\n \"The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing. Human beings encounter more ecology-climate-changing problems, including the zoonotic pathogens. We have to face some unknown pathogenic agents passively. To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown.\",\n \"To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown. This is especially true for infectious agents.\"\n]"},"document_id":{"kind":"number","value":2628,"string":"2,628"},"id":{"kind":"number","value":3847,"string":"3,847"}}},{"rowIdx":675,"cells":{"question":{"kind":"string","value":"What is a recent discovery?"},"answer":{"kind":"string","value":"Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host "},"context_chunks":{"kind":"list like","value":["nan Text: pecially with next-generation sequencing NGS for new virus genome discovery, e.g., Ruben Donis et al. sequenced a bat-derived influenza virus genome by using NGS in 2012, raising a serious question as to whether or not our seasonal or pandemic flu might have another reservoir host. Chen and colleagues confirmed the SFTSV independently by using NGS. Indeed, metagenomics analysis has yielded a great deal of new viruses, especially from the environment. Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal .","Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal . In this issue, six groups have been invited to present their recent findings on the emerging viruses, in addition to a previous report on H7N9 . Shi reviewed recent discoveries of new viruses or virus genomes from bat. Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 .","Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 . Recent MERS-CoV infection is another example for severe disease caused by used-to-be-less pathogenic coronaviruses. Shi and colleagues by using NGS have discovered many unknown animal viruses from bat, especially some important paramyxoviruses and reoviruses. Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city .","Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city . The potential roles of these viruses in bats for interspecies transmission are yet to be elucidated. Tan and colleagues specifically focused on the newly-emerged MERS-CoV. The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries.","The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries. In the initial diagnosis, the pan-coronavirus real-time reverse transcription polymerase chain reaction RT-PCR assay played a very important role for the identification of the causative agents. By using this method, scientists detected an expected-size PCR fragment for the corresponding conserved region of ORF1b of the replicase gene of a coronavirus. This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently .","This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently . Enterovirus has been known as serious human pathogens for a long time but their significance to the public health has been emphasized by the emergence of enterovirus 71 in 1998 as a serious pathogenic agents for children in Taiwan and re-emerged in mainland China in 2008 . In this issue, Duan and colleagues summarized the findings of new enteroviruses by using NGS. Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses.","Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses. The review also provided a detailed description of the NGS and related molecular methods for the virus discovery followed by a list of new enteroviruses found in human feces. These include viruses in the family of Piconaviridae, Parvoviridae, Circoviridae, Astroviridae and Polyomaviridae. Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future.","Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future. The disease caused by SFTSV, with a CFR of 12%, had been in China for a couple of years before the causative agent was finally identified. There are still a lot of questions remained unknown for this new virus and vigorous studies are in great need. The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts.","The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts. Therefore the effective control measures are still under evaluation. Vaccines protecting the SFTSV infection are under its way in Chinese Center for Disease Control and Prevention. Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered.","Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered. The new flavivirus, duck egg-drop syndrome virus DEDSV , is a good example. Su and colleagues reviewed the characterization of the DEDSV and its disease form in this issue. The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese .","The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese . Yet the transmission vector, though mosquitoes are suspected, has not been identified. Due to the public health concerns of its related viruses, potential human infection of DEDSV should be evaluated. Research on insect viruses is reviving in recent years. In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses.","In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses. Studies on these viruses can provide useful knowledge for our understanding about animal or human infecting viruses. More importantly, modification and application of insect-infecting viruses can be used as effective biologicals for the control of insect pest. The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing.","The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing. Human beings encounter more ecology-climate-changing problems, including the zoonotic pathogens. We have to face some unknown pathogenic agents passively. To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown.","To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown. This is especially true for infectious agents."],"string":"[\n \"nan Text: pecially with next-generation sequencing NGS for new virus genome discovery, e.g., Ruben Donis et al. sequenced a bat-derived influenza virus genome by using NGS in 2012, raising a serious question as to whether or not our seasonal or pandemic flu might have another reservoir host. Chen and colleagues confirmed the SFTSV independently by using NGS. Indeed, metagenomics analysis has yielded a great deal of new viruses, especially from the environment. Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal .\",\n \"Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal . In this issue, six groups have been invited to present their recent findings on the emerging viruses, in addition to a previous report on H7N9 . Shi reviewed recent discoveries of new viruses or virus genomes from bat. Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 .\",\n \"Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 . Recent MERS-CoV infection is another example for severe disease caused by used-to-be-less pathogenic coronaviruses. Shi and colleagues by using NGS have discovered many unknown animal viruses from bat, especially some important paramyxoviruses and reoviruses. Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city .\",\n \"Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city . The potential roles of these viruses in bats for interspecies transmission are yet to be elucidated. Tan and colleagues specifically focused on the newly-emerged MERS-CoV. The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries.\",\n \"The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries. In the initial diagnosis, the pan-coronavirus real-time reverse transcription polymerase chain reaction RT-PCR assay played a very important role for the identification of the causative agents. By using this method, scientists detected an expected-size PCR fragment for the corresponding conserved region of ORF1b of the replicase gene of a coronavirus. This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently .\",\n \"This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently . Enterovirus has been known as serious human pathogens for a long time but their significance to the public health has been emphasized by the emergence of enterovirus 71 in 1998 as a serious pathogenic agents for children in Taiwan and re-emerged in mainland China in 2008 . In this issue, Duan and colleagues summarized the findings of new enteroviruses by using NGS. Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses.\",\n \"Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses. The review also provided a detailed description of the NGS and related molecular methods for the virus discovery followed by a list of new enteroviruses found in human feces. These include viruses in the family of Piconaviridae, Parvoviridae, Circoviridae, Astroviridae and Polyomaviridae. Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future.\",\n \"Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future. The disease caused by SFTSV, with a CFR of 12%, had been in China for a couple of years before the causative agent was finally identified. There are still a lot of questions remained unknown for this new virus and vigorous studies are in great need. The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts.\",\n \"The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts. Therefore the effective control measures are still under evaluation. Vaccines protecting the SFTSV infection are under its way in Chinese Center for Disease Control and Prevention. Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered.\",\n \"Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered. The new flavivirus, duck egg-drop syndrome virus DEDSV , is a good example. Su and colleagues reviewed the characterization of the DEDSV and its disease form in this issue. The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese .\",\n \"The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese . Yet the transmission vector, though mosquitoes are suspected, has not been identified. Due to the public health concerns of its related viruses, potential human infection of DEDSV should be evaluated. Research on insect viruses is reviving in recent years. In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses.\",\n \"In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses. Studies on these viruses can provide useful knowledge for our understanding about animal or human infecting viruses. More importantly, modification and application of insect-infecting viruses can be used as effective biologicals for the control of insect pest. The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing.\",\n \"The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing. Human beings encounter more ecology-climate-changing problems, including the zoonotic pathogens. We have to face some unknown pathogenic agents passively. To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown.\",\n \"To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown. This is especially true for infectious agents.\"\n]"},"document_id":{"kind":"number","value":2628,"string":"2,628"},"id":{"kind":"number","value":3848,"string":"3,848"}}},{"rowIdx":676,"cells":{"question":{"kind":"string","value":"Which bat virus have been found to be linked with diseases?"},"answer":{"kind":"string","value":"potential human infecting HKU-1, 4, 5 and 9 . Recent MERS-CoV infection is another example for severe disease caused by used-to-be-less pathogenic coronaviruses. Shi and colleagues by using NGS have discovered many unknown animal viruses from bat, especially some important paramyxoviruses and reoviruses. Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses (with many genotypes, including rabies virus) in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city ."},"context_chunks":{"kind":"list like","value":["nan Text: pecially with next-generation sequencing NGS for new virus genome discovery, e.g., Ruben Donis et al. sequenced a bat-derived influenza virus genome by using NGS in 2012, raising a serious question as to whether or not our seasonal or pandemic flu might have another reservoir host. Chen and colleagues confirmed the SFTSV independently by using NGS. Indeed, metagenomics analysis has yielded a great deal of new viruses, especially from the environment. Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal .","Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal . In this issue, six groups have been invited to present their recent findings on the emerging viruses, in addition to a previous report on H7N9 . Shi reviewed recent discoveries of new viruses or virus genomes from bat. Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 .","Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 . Recent MERS-CoV infection is another example for severe disease caused by used-to-be-less pathogenic coronaviruses. Shi and colleagues by using NGS have discovered many unknown animal viruses from bat, especially some important paramyxoviruses and reoviruses. Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city .","Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city . The potential roles of these viruses in bats for interspecies transmission are yet to be elucidated. Tan and colleagues specifically focused on the newly-emerged MERS-CoV. The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries.","The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries. In the initial diagnosis, the pan-coronavirus real-time reverse transcription polymerase chain reaction RT-PCR assay played a very important role for the identification of the causative agents. By using this method, scientists detected an expected-size PCR fragment for the corresponding conserved region of ORF1b of the replicase gene of a coronavirus. This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently .","This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently . Enterovirus has been known as serious human pathogens for a long time but their significance to the public health has been emphasized by the emergence of enterovirus 71 in 1998 as a serious pathogenic agents for children in Taiwan and re-emerged in mainland China in 2008 . In this issue, Duan and colleagues summarized the findings of new enteroviruses by using NGS. Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses.","Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses. The review also provided a detailed description of the NGS and related molecular methods for the virus discovery followed by a list of new enteroviruses found in human feces. These include viruses in the family of Piconaviridae, Parvoviridae, Circoviridae, Astroviridae and Polyomaviridae. Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future.","Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future. The disease caused by SFTSV, with a CFR of 12%, had been in China for a couple of years before the causative agent was finally identified. There are still a lot of questions remained unknown for this new virus and vigorous studies are in great need. The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts.","The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts. Therefore the effective control measures are still under evaluation. Vaccines protecting the SFTSV infection are under its way in Chinese Center for Disease Control and Prevention. Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered.","Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered. The new flavivirus, duck egg-drop syndrome virus DEDSV , is a good example. Su and colleagues reviewed the characterization of the DEDSV and its disease form in this issue. The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese .","The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese . Yet the transmission vector, though mosquitoes are suspected, has not been identified. Due to the public health concerns of its related viruses, potential human infection of DEDSV should be evaluated. Research on insect viruses is reviving in recent years. In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses.","In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses. Studies on these viruses can provide useful knowledge for our understanding about animal or human infecting viruses. More importantly, modification and application of insect-infecting viruses can be used as effective biologicals for the control of insect pest. The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing.","The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing. Human beings encounter more ecology-climate-changing problems, including the zoonotic pathogens. We have to face some unknown pathogenic agents passively. To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown.","To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown. This is especially true for infectious agents."],"string":"[\n \"nan Text: pecially with next-generation sequencing NGS for new virus genome discovery, e.g., Ruben Donis et al. sequenced a bat-derived influenza virus genome by using NGS in 2012, raising a serious question as to whether or not our seasonal or pandemic flu might have another reservoir host. Chen and colleagues confirmed the SFTSV independently by using NGS. Indeed, metagenomics analysis has yielded a great deal of new viruses, especially from the environment. Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal .\",\n \"Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal . In this issue, six groups have been invited to present their recent findings on the emerging viruses, in addition to a previous report on H7N9 . Shi reviewed recent discoveries of new viruses or virus genomes from bat. Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 .\",\n \"Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 . Recent MERS-CoV infection is another example for severe disease caused by used-to-be-less pathogenic coronaviruses. Shi and colleagues by using NGS have discovered many unknown animal viruses from bat, especially some important paramyxoviruses and reoviruses. Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city .\",\n \"Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city . The potential roles of these viruses in bats for interspecies transmission are yet to be elucidated. Tan and colleagues specifically focused on the newly-emerged MERS-CoV. The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries.\",\n \"The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries. In the initial diagnosis, the pan-coronavirus real-time reverse transcription polymerase chain reaction RT-PCR assay played a very important role for the identification of the causative agents. By using this method, scientists detected an expected-size PCR fragment for the corresponding conserved region of ORF1b of the replicase gene of a coronavirus. This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently .\",\n \"This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently . Enterovirus has been known as serious human pathogens for a long time but their significance to the public health has been emphasized by the emergence of enterovirus 71 in 1998 as a serious pathogenic agents for children in Taiwan and re-emerged in mainland China in 2008 . In this issue, Duan and colleagues summarized the findings of new enteroviruses by using NGS. Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses.\",\n \"Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses. The review also provided a detailed description of the NGS and related molecular methods for the virus discovery followed by a list of new enteroviruses found in human feces. These include viruses in the family of Piconaviridae, Parvoviridae, Circoviridae, Astroviridae and Polyomaviridae. Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future.\",\n \"Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future. The disease caused by SFTSV, with a CFR of 12%, had been in China for a couple of years before the causative agent was finally identified. There are still a lot of questions remained unknown for this new virus and vigorous studies are in great need. The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts.\",\n \"The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts. Therefore the effective control measures are still under evaluation. Vaccines protecting the SFTSV infection are under its way in Chinese Center for Disease Control and Prevention. Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered.\",\n \"Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered. The new flavivirus, duck egg-drop syndrome virus DEDSV , is a good example. Su and colleagues reviewed the characterization of the DEDSV and its disease form in this issue. The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese .\",\n \"The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese . Yet the transmission vector, though mosquitoes are suspected, has not been identified. Due to the public health concerns of its related viruses, potential human infection of DEDSV should be evaluated. Research on insect viruses is reviving in recent years. In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses.\",\n \"In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses. Studies on these viruses can provide useful knowledge for our understanding about animal or human infecting viruses. More importantly, modification and application of insect-infecting viruses can be used as effective biologicals for the control of insect pest. The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing.\",\n \"The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing. Human beings encounter more ecology-climate-changing problems, including the zoonotic pathogens. We have to face some unknown pathogenic agents passively. To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown.\",\n \"To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown. This is especially true for infectious agents.\"\n]"},"document_id":{"kind":"number","value":2628,"string":"2,628"},"id":{"kind":"number","value":3849,"string":"3,849"}}},{"rowIdx":677,"cells":{"question":{"kind":"string","value":"What assay played an important role?"},"answer":{"kind":"string","value":"reverse transcription polymerase chain reaction (RT-PCR)"},"context_chunks":{"kind":"list like","value":["nan Text: pecially with next-generation sequencing NGS for new virus genome discovery, e.g., Ruben Donis et al. sequenced a bat-derived influenza virus genome by using NGS in 2012, raising a serious question as to whether or not our seasonal or pandemic flu might have another reservoir host. Chen and colleagues confirmed the SFTSV independently by using NGS. Indeed, metagenomics analysis has yielded a great deal of new viruses, especially from the environment. Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal .","Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal . In this issue, six groups have been invited to present their recent findings on the emerging viruses, in addition to a previous report on H7N9 . Shi reviewed recent discoveries of new viruses or virus genomes from bat. Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 .","Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 . Recent MERS-CoV infection is another example for severe disease caused by used-to-be-less pathogenic coronaviruses. Shi and colleagues by using NGS have discovered many unknown animal viruses from bat, especially some important paramyxoviruses and reoviruses. Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city .","Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city . The potential roles of these viruses in bats for interspecies transmission are yet to be elucidated. Tan and colleagues specifically focused on the newly-emerged MERS-CoV. The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries.","The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries. In the initial diagnosis, the pan-coronavirus real-time reverse transcription polymerase chain reaction RT-PCR assay played a very important role for the identification of the causative agents. By using this method, scientists detected an expected-size PCR fragment for the corresponding conserved region of ORF1b of the replicase gene of a coronavirus. This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently .","This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently . Enterovirus has been known as serious human pathogens for a long time but their significance to the public health has been emphasized by the emergence of enterovirus 71 in 1998 as a serious pathogenic agents for children in Taiwan and re-emerged in mainland China in 2008 . In this issue, Duan and colleagues summarized the findings of new enteroviruses by using NGS. Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses.","Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses. The review also provided a detailed description of the NGS and related molecular methods for the virus discovery followed by a list of new enteroviruses found in human feces. These include viruses in the family of Piconaviridae, Parvoviridae, Circoviridae, Astroviridae and Polyomaviridae. Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future.","Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future. The disease caused by SFTSV, with a CFR of 12%, had been in China for a couple of years before the causative agent was finally identified. There are still a lot of questions remained unknown for this new virus and vigorous studies are in great need. The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts.","The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts. Therefore the effective control measures are still under evaluation. Vaccines protecting the SFTSV infection are under its way in Chinese Center for Disease Control and Prevention. Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered.","Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered. The new flavivirus, duck egg-drop syndrome virus DEDSV , is a good example. Su and colleagues reviewed the characterization of the DEDSV and its disease form in this issue. The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese .","The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese . Yet the transmission vector, though mosquitoes are suspected, has not been identified. Due to the public health concerns of its related viruses, potential human infection of DEDSV should be evaluated. Research on insect viruses is reviving in recent years. In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses.","In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses. Studies on these viruses can provide useful knowledge for our understanding about animal or human infecting viruses. More importantly, modification and application of insect-infecting viruses can be used as effective biologicals for the control of insect pest. The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing.","The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing. Human beings encounter more ecology-climate-changing problems, including the zoonotic pathogens. We have to face some unknown pathogenic agents passively. To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown.","To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown. This is especially true for infectious agents."],"string":"[\n \"nan Text: pecially with next-generation sequencing NGS for new virus genome discovery, e.g., Ruben Donis et al. sequenced a bat-derived influenza virus genome by using NGS in 2012, raising a serious question as to whether or not our seasonal or pandemic flu might have another reservoir host. Chen and colleagues confirmed the SFTSV independently by using NGS. Indeed, metagenomics analysis has yielded a great deal of new viruses, especially from the environment. Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal .\",\n \"Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal . In this issue, six groups have been invited to present their recent findings on the emerging viruses, in addition to a previous report on H7N9 . Shi reviewed recent discoveries of new viruses or virus genomes from bat. Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 .\",\n \"Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 . Recent MERS-CoV infection is another example for severe disease caused by used-to-be-less pathogenic coronaviruses. Shi and colleagues by using NGS have discovered many unknown animal viruses from bat, especially some important paramyxoviruses and reoviruses. Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city .\",\n \"Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city . The potential roles of these viruses in bats for interspecies transmission are yet to be elucidated. Tan and colleagues specifically focused on the newly-emerged MERS-CoV. The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries.\",\n \"The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries. In the initial diagnosis, the pan-coronavirus real-time reverse transcription polymerase chain reaction RT-PCR assay played a very important role for the identification of the causative agents. By using this method, scientists detected an expected-size PCR fragment for the corresponding conserved region of ORF1b of the replicase gene of a coronavirus. This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently .\",\n \"This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently . Enterovirus has been known as serious human pathogens for a long time but their significance to the public health has been emphasized by the emergence of enterovirus 71 in 1998 as a serious pathogenic agents for children in Taiwan and re-emerged in mainland China in 2008 . In this issue, Duan and colleagues summarized the findings of new enteroviruses by using NGS. Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses.\",\n \"Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses. The review also provided a detailed description of the NGS and related molecular methods for the virus discovery followed by a list of new enteroviruses found in human feces. These include viruses in the family of Piconaviridae, Parvoviridae, Circoviridae, Astroviridae and Polyomaviridae. Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future.\",\n \"Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future. The disease caused by SFTSV, with a CFR of 12%, had been in China for a couple of years before the causative agent was finally identified. There are still a lot of questions remained unknown for this new virus and vigorous studies are in great need. The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts.\",\n \"The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts. Therefore the effective control measures are still under evaluation. Vaccines protecting the SFTSV infection are under its way in Chinese Center for Disease Control and Prevention. Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered.\",\n \"Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered. The new flavivirus, duck egg-drop syndrome virus DEDSV , is a good example. Su and colleagues reviewed the characterization of the DEDSV and its disease form in this issue. The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese .\",\n \"The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese . Yet the transmission vector, though mosquitoes are suspected, has not been identified. Due to the public health concerns of its related viruses, potential human infection of DEDSV should be evaluated. Research on insect viruses is reviving in recent years. In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses.\",\n \"In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses. Studies on these viruses can provide useful knowledge for our understanding about animal or human infecting viruses. More importantly, modification and application of insect-infecting viruses can be used as effective biologicals for the control of insect pest. The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing.\",\n \"The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing. Human beings encounter more ecology-climate-changing problems, including the zoonotic pathogens. We have to face some unknown pathogenic agents passively. To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown.\",\n \"To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown. This is especially true for infectious agents.\"\n]"},"document_id":{"kind":"number","value":2628,"string":"2,628"},"id":{"kind":"number","value":3850,"string":"3,850"}}},{"rowIdx":678,"cells":{"question":{"kind":"string","value":"What was the death toll in the 1918-1919 Spanish Influenza epidemic?"},"answer":{"kind":"string","value":"50 million deaths worldwide"},"context_chunks":{"kind":"list like","value":["1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.","But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..","Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.","The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.","Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.","But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .","Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.","No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .","These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.","The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .","Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.","Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .","The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.","Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.","Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.","In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.","The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.","The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.","However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?","Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.","And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.","As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.","Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.","Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.","One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.","Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?","One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?","Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.","Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..","The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.","Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.","These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.","The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.","The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .","The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.","But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.","But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.","Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.","In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.","In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.","Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.","Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.","There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.","Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.","Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.","His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. 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Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.","J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.","J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.","Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."],"string":"[\n \"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\\\" and David M. Morens1- The “Spanish\\\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\",\n \"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..\",\n \"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.\",\n \"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.\",\n \"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.\",\n \"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .\",\n \"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.\",\n \"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .\",\n \"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.\",\n \"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .\",\n \"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.\",\n \"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .\",\n \"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.\",\n \"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.\",\n \"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.\",\n \"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.\",\n \"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.\",\n \"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.\",\n \"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?\",\n \"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.\",\n \"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.\",\n \"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.\",\n \"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.\",\n \"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.\",\n \"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.\",\n \"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?\",\n \"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\",\n \"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.\",\n \"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..\",\n \"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.\",\n \"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.\",\n \"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.\",\n \"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.\",\n \"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .\",\n \"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.\",\n \"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.\",\n \"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.\",\n \"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.\",\n \"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.\",\n \"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.\",\n \"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\",\n \"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.\",\n \"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\",\n \"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.\",\n \"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.\",\n \"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.\",\n \"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.\",\n \"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.\",\n \"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.\",\n \"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.\",\n \"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.\",\n \"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.\",\n \"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.\",\n \"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.\",\n \"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.\",\n \"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.\",\n \"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.\",\n \"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.\",\n \"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.\",\n \"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.\"\n]"},"document_id":{"kind":"number","value":2684,"string":"2,684"},"id":{"kind":"number","value":1057,"string":"1,057"}}},{"rowIdx":679,"cells":{"question":{"kind":"string","value":"How many people were infected during the 1918 Spanish Influenza epidemic?"},"answer":{"kind":"string","value":"An estimated one third of the world’s population (or\nz500 million persons) were infected and had clinical-\nly apparent illnesses"},"context_chunks":{"kind":"list like","value":["1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.","But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..","Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.","The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.","Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.","But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .","Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.","No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .","These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.","The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .","Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.","Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .","The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.","Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.","Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.","In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.","The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.","The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.","However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?","Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.","And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.","As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.","Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.","Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.","One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.","Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?","One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?","Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.","Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..","The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.","Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.","These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.","The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.","The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .","The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.","But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.","But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.","Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.","In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.","In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.","Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.","Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.","There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.","Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.","Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.","His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.","Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.","Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.","Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.","12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.","J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.","Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.","Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.","Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.","Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.","Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.","A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.","J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.","J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.","Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."],"string":"[\n \"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\\\" and David M. Morens1- The “Spanish\\\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\",\n \"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..\",\n \"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.\",\n \"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.\",\n \"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.\",\n \"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .\",\n \"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.\",\n \"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .\",\n \"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.\",\n \"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .\",\n \"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.\",\n \"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .\",\n \"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.\",\n \"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.\",\n \"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.\",\n \"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.\",\n \"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.\",\n \"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.\",\n \"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?\",\n \"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.\",\n \"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.\",\n \"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.\",\n \"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.\",\n \"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.\",\n \"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.\",\n \"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?\",\n \"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\",\n \"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.\",\n \"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..\",\n \"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.\",\n \"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.\",\n \"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.\",\n \"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.\",\n \"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .\",\n \"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.\",\n \"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.\",\n \"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.\",\n \"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.\",\n \"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.\",\n \"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.\",\n \"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\",\n \"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.\",\n \"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\",\n \"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.\",\n \"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.\",\n \"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.\",\n \"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.\",\n \"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.\",\n \"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.\",\n \"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.\",\n \"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.\",\n \"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.\",\n \"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.\",\n \"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.\",\n \"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.\",\n \"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.\",\n \"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.\",\n \"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.\",\n \"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.\",\n \"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.\"\n]"},"document_id":{"kind":"number","value":2684,"string":"2,684"},"id":{"kind":"number","value":1058,"string":"1,058"}}},{"rowIdx":680,"cells":{"question":{"kind":"string","value":"What was the case fatality rate in the 1918 Spanish Influenza epidemic?"},"answer":{"kind":"string","value":"Case-\nfatality rates were >2.5%, compared to <0.1% in other\ninfluenza pandemics"},"context_chunks":{"kind":"list like","value":["1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.","But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..","Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.","The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.","Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.","But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .","Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.","No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .","These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.","The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .","Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.","Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .","The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.","Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.","Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.","In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.","The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.","The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.","However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?","Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.","And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.","As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.","Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.","Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.","One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.","Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?","One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?","Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.","Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..","The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.","Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.","These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.","The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.","The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .","The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.","But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.","But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.","Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.","In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.","In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.","Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.","Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.","There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.","Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.","Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.","His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.","Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.","Johnson NPAS, Mueller J. 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Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.","12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.","J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.","Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.","Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.","Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.","Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.","Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.","A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.","J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.","J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.","Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."],"string":"[\n \"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\\\" and David M. Morens1- The “Spanish\\\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\",\n \"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..\",\n \"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.\",\n \"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.\",\n \"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.\",\n \"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .\",\n \"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.\",\n \"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .\",\n \"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.\",\n \"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .\",\n \"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.\",\n \"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .\",\n \"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.\",\n \"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.\",\n \"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.\",\n \"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.\",\n \"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.\",\n \"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.\",\n \"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?\",\n \"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.\",\n \"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.\",\n \"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.\",\n \"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.\",\n \"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.\",\n \"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.\",\n \"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?\",\n \"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\",\n \"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.\",\n \"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..\",\n \"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.\",\n \"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.\",\n \"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.\",\n \"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.\",\n \"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .\",\n \"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.\",\n \"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.\",\n \"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.\",\n \"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.\",\n \"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.\",\n \"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.\",\n \"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\",\n \"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.\",\n \"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\",\n \"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.\",\n \"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.\",\n \"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. 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Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.\",\n \"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.\",\n \"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.\",\n \"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.\",\n \"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.\",\n \"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.\",\n \"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.\",\n \"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.\",\n \"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.\"\n]"},"document_id":{"kind":"number","value":2684,"string":"2,684"},"id":{"kind":"number","value":1059,"string":"1,059"}}},{"rowIdx":681,"cells":{"question":{"kind":"string","value":"What was the death toll in the 1918-1919 Spanish Influenza epidemic?"},"answer":{"kind":"string","value":"Total deaths were estimated at\nz50 million (577) and were arguably as high as 100 mil-\nlion "},"context_chunks":{"kind":"list like","value":["1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.","But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..","Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.","The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.","Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.","But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .","Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.","No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .","These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.","The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .","Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.","Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .","The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.","Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.","Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.","In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.","The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.","The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.","However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?","Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.","And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.","As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.","Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.","Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.","One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.","Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?","One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?","Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.","Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..","The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.","Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.","These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.","The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.","The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .","The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.","But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.","But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.","Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.","In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.","In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.","Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.","Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.","There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.","Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.","Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.","His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.","Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.","Johnson NPAS, Mueller J. 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Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.","12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.","J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.","Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.","Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.","Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. 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Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.","A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.","J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.","J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.","Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."],"string":"[\n \"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\\\" and David M. Morens1- The “Spanish\\\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\",\n \"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..\",\n \"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.\",\n \"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.\",\n \"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.\",\n \"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .\",\n \"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.\",\n \"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .\",\n \"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.\",\n \"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .\",\n \"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.\",\n \"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .\",\n \"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.\",\n \"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.\",\n \"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.\",\n \"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.\",\n \"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.\",\n \"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.\",\n \"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?\",\n \"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.\",\n \"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.\",\n \"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.\",\n \"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.\",\n \"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.\",\n \"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.\",\n \"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?\",\n \"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\",\n \"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.\",\n \"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..\",\n \"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.\",\n \"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.\",\n \"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.\",\n \"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.\",\n \"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .\",\n \"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.\",\n \"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.\",\n \"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.\",\n \"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.\",\n \"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.\",\n \"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.\",\n \"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\",\n \"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.\",\n \"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\",\n \"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.\",\n \"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.\",\n \"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.\",\n \"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.\",\n \"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.\",\n \"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.\",\n \"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.\",\n \"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.\",\n \"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.\",\n \"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.\",\n \"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.\",\n \"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.\",\n \"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.\",\n \"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.\",\n \"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.\",\n \"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.\",\n \"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.\"\n]"},"document_id":{"kind":"number","value":2684,"string":"2,684"},"id":{"kind":"number","value":1060,"string":"1,060"}}},{"rowIdx":682,"cells":{"question":{"kind":"string","value":"Are the modern day Influenza viruses related to the 1918 Spanish Influenza virus?"},"answer":{"kind":"string","value":" All influenza A pandemics since that time, and\nindeed almost all cases of influenza A worldwide (except-\ning human infections from avian Viruses such as H5N1 and\nH7N7), have been caused by descendants of the 1918\nVirus, including “drifted” H1N1 Viruses and reassorted\nH2N2 and H3N2 Viruses."},"context_chunks":{"kind":"list like","value":["1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.","But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..","Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.","The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.","Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.","But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .","Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.","No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .","These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.","The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .","Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.","Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .","The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.","Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.","Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.","In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.","The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.","The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.","However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?","Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.","And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.","As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.","Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.","Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.","One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.","Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?","One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?","Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.","Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..","The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.","Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.","These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.","The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.","The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .","The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.","But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.","But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.","Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.","In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.","In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.","Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.","Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.","There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.","Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.","Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.","His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. 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Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.","Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."],"string":"[\n \"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\\\" and David M. Morens1- The “Spanish\\\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\",\n \"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..\",\n \"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.\",\n \"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.\",\n \"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.\",\n \"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .\",\n \"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.\",\n \"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .\",\n \"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.\",\n \"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .\",\n \"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.\",\n \"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .\",\n \"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.\",\n \"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.\",\n \"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.\",\n \"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.\",\n \"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.\",\n \"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.\",\n \"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?\",\n \"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.\",\n \"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.\",\n \"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.\",\n \"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.\",\n \"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.\",\n \"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.\",\n \"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?\",\n \"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\",\n \"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.\",\n \"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..\",\n \"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.\",\n \"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.\",\n \"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.\",\n \"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.\",\n \"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .\",\n \"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.\",\n \"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.\",\n \"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.\",\n \"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.\",\n \"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.\",\n \"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.\",\n \"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\",\n \"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.\",\n \"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\",\n \"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.\",\n \"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.\",\n \"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.\",\n \"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.\",\n \"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.\",\n \"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.\",\n \"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.\",\n \"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.\",\n \"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.\",\n \"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.\",\n \"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.\",\n \"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.\",\n \"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.\",\n \"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.\",\n \"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.\",\n \"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.\",\n \"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.\"\n]"},"document_id":{"kind":"number","value":2684,"string":"2,684"},"id":{"kind":"number","value":1062,"string":"1,062"}}},{"rowIdx":683,"cells":{"question":{"kind":"string","value":"Why is the Spanish Influenza virus the Mother of the modern influenza viruses?"},"answer":{"kind":"string","value":"The latter are composed of key\ngenes from the 1918 Virus, updated by subsequently-incor—\nporated avian influenza genes that code for novel surface\n\n \n\n*Armed Forces Institute of Pathology, Rockville, Maryland, USA;\nand TNational Institutes of Health, Bethesda, Maryland, USA\n\nproteins, making the 1918 Virus indeed the “mother” of all\npandemics."},"context_chunks":{"kind":"list like","value":["1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.","But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..","Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.","The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.","Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.","But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .","Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.","No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .","These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.","The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .","Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.","Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .","The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.","Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.","Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.","In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.","The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.","The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.","However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?","Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.","And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.","As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.","Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.","Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.","One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.","Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?","One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?","Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.","Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..","The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.","Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.","These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.","The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.","The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .","The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.","But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.","But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.","Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.","In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.","In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.","Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.","Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.","There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.","Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.","Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.","His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. 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Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.","Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.","Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.","Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.","A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.","J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.","J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.","Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."],"string":"[\n \"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\\\" and David M. Morens1- The “Spanish\\\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\",\n \"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..\",\n \"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.\",\n \"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.\",\n \"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.\",\n \"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .\",\n \"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.\",\n \"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .\",\n \"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.\",\n \"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .\",\n \"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.\",\n \"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .\",\n \"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.\",\n \"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.\",\n \"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.\",\n \"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.\",\n \"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.\",\n \"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.\",\n \"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?\",\n \"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.\",\n \"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.\",\n \"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.\",\n \"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.\",\n \"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.\",\n \"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.\",\n \"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?\",\n \"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\",\n \"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.\",\n \"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..\",\n \"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.\",\n \"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.\",\n \"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.\",\n \"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.\",\n \"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .\",\n \"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.\",\n \"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.\",\n \"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.\",\n \"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.\",\n \"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.\",\n \"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.\",\n \"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\",\n \"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.\",\n \"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\",\n \"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.\",\n \"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.\",\n \"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.\",\n \"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.\",\n \"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.\",\n \"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.\",\n \"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.\",\n \"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.\",\n \"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.\",\n \"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.\",\n \"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.\",\n \"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.\",\n \"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.\",\n \"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.\",\n \"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.\",\n \"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.\",\n \"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.\"\n]"},"document_id":{"kind":"number","value":2684,"string":"2,684"},"id":{"kind":"number","value":1063,"string":"1,063"}}},{"rowIdx":684,"cells":{"question":{"kind":"string","value":"When was it determined that the 1918 pandemic was caused by the H1N1 Influenza virus?"},"answer":{"kind":"string","value":"That question did not begin to be resolved until the 1930s,\nwhen closely related influenza Viruses (now known to be\nH1N1 Viruses) were isolated, first from pigs and shortly\nthereafter from humans. Seroepidemiologic studies soon\nlinked both of these viruses to the 1918 pandemic"},"context_chunks":{"kind":"list like","value":["1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.","But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..","Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.","The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.","Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.","But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .","Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.","No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .","These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.","The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .","Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.","Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .","The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.","Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.","Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.","In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.","The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.","The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.","However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?","Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.","And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.","As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.","Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.","Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.","One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.","Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?","One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?","Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.","Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..","The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.","Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.","These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.","The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.","The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .","The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.","But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.","But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.","Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.","In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.","In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.","Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.","Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.","There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.","Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.","Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.","His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.","Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.","Johnson NPAS, Mueller J. 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Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.","Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.","Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.","Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.","Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.","Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.","A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.","J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.","J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.","Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."],"string":"[\n \"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\\\" and David M. Morens1- The “Spanish\\\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\",\n \"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..\",\n \"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.\",\n \"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.\",\n \"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.\",\n \"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .\",\n \"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.\",\n \"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .\",\n \"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.\",\n \"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .\",\n \"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.\",\n \"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .\",\n \"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.\",\n \"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.\",\n \"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.\",\n \"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.\",\n \"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.\",\n \"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.\",\n \"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?\",\n \"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.\",\n \"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.\",\n \"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.\",\n \"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.\",\n \"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.\",\n \"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.\",\n \"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?\",\n \"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\",\n \"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.\",\n \"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..\",\n \"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.\",\n \"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.\",\n \"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.\",\n \"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.\",\n \"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .\",\n \"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.\",\n \"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.\",\n \"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.\",\n \"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.\",\n \"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.\",\n \"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.\",\n \"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\",\n \"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.\",\n \"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\",\n \"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.\",\n \"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.\",\n \"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. 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Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.\",\n \"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.\",\n \"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.\",\n \"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.\"\n]"},"document_id":{"kind":"number","value":2684,"string":"2,684"},"id":{"kind":"number","value":1064,"string":"1,064"}}},{"rowIdx":685,"cells":{"question":{"kind":"string","value":"Did the Spanish Influenza or Swine flu or the H1N1 virus disappear in humans for some time?"},"answer":{"kind":"string","value":" descendants of the 1918\nVirus still persists enzootically in pigs. They probably also\ncirculated continuously in humans, undergoing gradual\nantigenic drift and causing annual epidemics, until the\n1950s. With the appearance of a new H2N2 pandemic\nstrain in 1957 (“Asian flu”), the direct H1N1 Viral descen-\ndants 0f the 1918 pandemic strain disappeared from human\ncirculation entirely, although the related lineage persisted\nenzootically in pigs."},"context_chunks":{"kind":"list like","value":["1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.","But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..","Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.","The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.","Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.","But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .","Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.","No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .","These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.","The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .","Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.","Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .","The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.","Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.","Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.","In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.","The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.","The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.","However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?","Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.","And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.","As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.","Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.","Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.","One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.","Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?","One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?","Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.","Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..","The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.","Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.","These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.","The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.","The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .","The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.","But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.","But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.","Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.","In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.","In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.","Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.","Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.","There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.","Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.","Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.","His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.","Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.","Johnson NPAS, Mueller J. 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Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.","12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.","J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.","Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.","Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.","Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.","Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.","Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.","A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.","J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.","J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.","Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."],"string":"[\n \"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\\\" and David M. Morens1- The “Spanish\\\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\",\n \"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..\",\n \"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.\",\n \"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.\",\n \"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.\",\n \"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .\",\n \"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.\",\n \"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .\",\n \"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.\",\n \"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .\",\n \"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.\",\n \"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .\",\n \"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.\",\n \"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.\",\n \"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.\",\n \"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.\",\n \"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.\",\n \"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.\",\n \"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?\",\n \"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.\",\n \"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.\",\n \"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.\",\n \"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.\",\n \"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.\",\n \"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.\",\n \"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?\",\n \"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\",\n \"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.\",\n \"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..\",\n \"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.\",\n \"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.\",\n \"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.\",\n \"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.\",\n \"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .\",\n \"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.\",\n \"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.\",\n \"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.\",\n \"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.\",\n \"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.\",\n \"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.\",\n \"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\",\n \"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.\",\n \"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\",\n \"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.\",\n \"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.\",\n \"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. 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Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.\",\n \"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.\",\n \"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.\",\n \"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.\",\n \"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.\",\n \"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.\",\n \"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.\",\n \"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.\",\n \"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.\",\n \"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.\",\n \"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.\",\n \"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.\"\n]"},"document_id":{"kind":"number","value":2684,"string":"2,684"},"id":{"kind":"number","value":1065,"string":"1,065"}}},{"rowIdx":686,"cells":{"question":{"kind":"string","value":"When did the Swine Flu (Spanish Influenza) virus reappear in humans?"},"answer":{"kind":"string","value":" But in 1977, human H1N1 Viruses\nsuddenly “reemerged” from a laboratory freezer (9). They\ncontinue to circulate endemically and epidemically."},"context_chunks":{"kind":"list like","value":["1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.","But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..","Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.","The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.","Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.","But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .","Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.","No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .","These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.","The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .","Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.","Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .","The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.","Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.","Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.","In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.","The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.","The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.","However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?","Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.","And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.","As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.","Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.","Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.","One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.","Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?","One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?","Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.","Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..","The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.","Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.","These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.","The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.","The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .","The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.","But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.","But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.","Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.","In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.","In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.","Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.","Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.","There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.","Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.","Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.","His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.","Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.","Johnson NPAS, Mueller J. 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Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.","Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."],"string":"[\n \"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\\\" and David M. Morens1- The “Spanish\\\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\",\n \"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..\",\n \"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.\",\n \"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.\",\n \"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.\",\n \"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .\",\n \"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.\",\n \"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .\",\n \"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.\",\n \"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .\",\n \"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.\",\n \"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .\",\n \"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.\",\n \"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.\",\n \"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.\",\n \"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.\",\n \"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.\",\n \"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.\",\n \"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?\",\n \"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.\",\n \"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.\",\n \"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.\",\n \"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.\",\n \"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.\",\n \"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.\",\n \"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?\",\n \"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\",\n \"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.\",\n \"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..\",\n \"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.\",\n \"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.\",\n \"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.\",\n \"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.\",\n \"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .\",\n \"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.\",\n \"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.\",\n \"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.\",\n \"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.\",\n \"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.\",\n \"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.\",\n \"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\",\n \"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.\",\n \"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\",\n \"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.\",\n \"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.\",\n \"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.\",\n \"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.\",\n \"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.\",\n \"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.\",\n \"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.\",\n \"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.\",\n \"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.\",\n \"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.\",\n \"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.\",\n \"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.\",\n \"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.\",\n \"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.\",\n \"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.\",\n \"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.\",\n \"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.\"\n]"},"document_id":{"kind":"number","value":2684,"string":"2,684"},"id":{"kind":"number","value":1066,"string":"1,066"}}},{"rowIdx":687,"cells":{"question":{"kind":"string","value":"What descendant lineages of the swine flu (Spanish Influenza) virus were identified in 2006?"},"answer":{"kind":"string","value":" 2 major descendant lineages of the 1918\nH1N1 Virus, as well as 2 additional reassortant lineages,\npersist naturally: a human epidemic/endemic H1N1 line-\nage, a porcine enzootic H1N1 lineage (so-called classic\nswine flu), and the reassorted human H3N2 Virus lineage,\nwhich like the human H1N1 Virus, has led to a porcine\nH3N2 lineage."},"context_chunks":{"kind":"list like","value":["1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.","But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..","Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.","The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.","Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.","But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .","Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.","No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .","These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.","The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .","Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.","Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .","The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.","Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.","Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.","In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.","The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.","The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.","However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?","Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.","And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.","As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.","Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.","Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.","One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.","Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?","One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?","Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.","Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..","The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.","Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.","These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.","The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.","The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .","The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.","But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.","But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.","Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.","In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.","In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.","Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.","Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.","There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.","Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.","Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.","His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. 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Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.","J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.","J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.","Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."],"string":"[\n \"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\\\" and David M. Morens1- The “Spanish\\\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\",\n \"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..\",\n \"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.\",\n \"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.\",\n \"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.\",\n \"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .\",\n \"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.\",\n \"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .\",\n \"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.\",\n \"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .\",\n \"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.\",\n \"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .\",\n \"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.\",\n \"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.\",\n \"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.\",\n \"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.\",\n \"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.\",\n \"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.\",\n \"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?\",\n \"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.\",\n \"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.\",\n \"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.\",\n \"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.\",\n \"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.\",\n \"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.\",\n \"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?\",\n \"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\",\n \"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.\",\n \"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..\",\n \"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.\",\n \"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.\",\n \"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.\",\n \"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.\",\n \"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .\",\n \"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.\",\n \"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.\",\n \"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.\",\n \"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.\",\n \"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.\",\n \"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.\",\n \"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\",\n \"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.\",\n \"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\",\n \"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.\",\n \"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.\",\n \"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.\",\n \"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.\",\n \"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.\",\n \"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.\",\n \"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.\",\n \"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.\",\n \"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.\",\n \"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.\",\n \"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.\",\n \"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.\",\n \"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.\",\n \"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.\",\n \"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.\",\n \"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.\",\n \"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.\"\n]"},"document_id":{"kind":"number","value":2684,"string":"2,684"},"id":{"kind":"number","value":1068,"string":"1,068"}}},{"rowIdx":688,"cells":{"question":{"kind":"string","value":"Are the modern descendant influenza viruses as dangerous as the 1918 parent swine flu (Spanish Influenza) H1N1 virus?"},"answer":{"kind":"string","value":"None of these Viral descendants, however,\napproaches the pathogenicity of the 1918 parent Virus."},"context_chunks":{"kind":"list like","value":["1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.","But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..","Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.","The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.","Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.","But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .","Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.","No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .","These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.","The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .","Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.","Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .","The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.","Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.","Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.","In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.","The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.","The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.","However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?","Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.","And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.","As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.","Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.","Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.","One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.","Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?","One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?","Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.","Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..","The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.","Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.","These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.","The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.","The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .","The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.","But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.","But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.","Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.","In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.","In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.","Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.","Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.","There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.","Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.","Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.","His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.","Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.","Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.","Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.","12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.","J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.","Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.","Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.","Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.","Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.","Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.","A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.","J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.","J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.","Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."],"string":"[\n \"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\\\" and David M. Morens1- The “Spanish\\\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\",\n \"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..\",\n \"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.\",\n \"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.\",\n \"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.\",\n \"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .\",\n \"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.\",\n \"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .\",\n \"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.\",\n \"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .\",\n \"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.\",\n \"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .\",\n \"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.\",\n \"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.\",\n \"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.\",\n \"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.\",\n \"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.\",\n \"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.\",\n \"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?\",\n \"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.\",\n \"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.\",\n \"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.\",\n \"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.\",\n \"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.\",\n \"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.\",\n \"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?\",\n \"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\",\n \"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.\",\n \"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..\",\n \"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.\",\n \"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.\",\n \"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.\",\n \"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.\",\n \"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .\",\n \"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.\",\n \"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.\",\n \"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.\",\n \"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.\",\n \"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.\",\n \"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.\",\n \"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\",\n \"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.\",\n \"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\",\n \"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.\",\n \"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.\",\n \"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.\",\n \"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.\",\n \"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.\",\n \"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.\",\n \"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.\",\n \"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.\",\n \"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.\",\n \"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.\",\n \"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.\",\n \"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.\",\n \"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.\",\n \"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.\",\n \"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.\",\n \"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.\",\n \"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.\"\n]"},"document_id":{"kind":"number","value":2684,"string":"2,684"},"id":{"kind":"number","value":1070,"string":"1,070"}}},{"rowIdx":689,"cells":{"question":{"kind":"string","value":"How dangerous are the modern H1N1 (swine flu) and the H3N2 (Influenza A) viruses compared to the 1918 H1N1 (swine flu Spanish Influenza) viruses?"},"answer":{"kind":"string","value":"the human H1N1 and H3N2 lin-\neages have both been associated with substantially lower\nrates ofillness and death than the virus of 1918. In fact, cur-\nrent H1N1 death rates are even lower than those for H3N2\nlineage strains (prevalent from 1968 until the present)."},"context_chunks":{"kind":"list like","value":["1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.","But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..","Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.","The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.","Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.","But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .","Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.","No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .","These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.","The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .","Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.","Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .","The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.","Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.","Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.","In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.","The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.","The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.","However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?","Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.","And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.","As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.","Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.","Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.","One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.","Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?","One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?","Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.","Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..","The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.","Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.","These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.","The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.","The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .","The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.","But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.","But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.","Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.","In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.","In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.","Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.","Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.","There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.","Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.","Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.","His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.","Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.","Johnson NPAS, Mueller J. 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Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.","12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.","J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.","Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.","Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.","Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.","Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.","Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.","A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.","J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.","J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.","Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."],"string":"[\n \"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\\\" and David M. Morens1- The “Spanish\\\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\",\n \"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..\",\n \"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.\",\n \"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.\",\n \"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.\",\n \"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .\",\n \"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.\",\n \"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .\",\n \"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.\",\n \"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .\",\n \"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.\",\n \"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .\",\n \"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.\",\n \"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.\",\n \"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.\",\n \"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.\",\n \"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.\",\n \"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.\",\n \"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?\",\n \"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.\",\n \"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.\",\n \"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.\",\n \"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.\",\n \"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.\",\n \"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.\",\n \"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?\",\n \"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\",\n \"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.\",\n \"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..\",\n \"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.\",\n \"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.\",\n \"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.\",\n \"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.\",\n \"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .\",\n \"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.\",\n \"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.\",\n \"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.\",\n \"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.\",\n \"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.\",\n \"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.\",\n \"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\",\n \"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.\",\n \"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\",\n \"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.\",\n \"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.\",\n \"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.\",\n \"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.\",\n \"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.\",\n \"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.\",\n \"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.\",\n \"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.\",\n \"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.\",\n \"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.\",\n \"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.\",\n \"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.\",\n \"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.\",\n \"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.\",\n \"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.\",\n \"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.\",\n \"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.\"\n]"},"document_id":{"kind":"number","value":2684,"string":"2,684"},"id":{"kind":"number","value":1072,"string":"1,072"}}},{"rowIdx":690,"cells":{"question":{"kind":"string","value":"Are the descendant H1N1 strains of the 1918 H1N1 swine flu (Spanish Influenza) virus, still prevalent?"},"answer":{"kind":"string","value":"H1N1 Viruses descended from the 1918 strain, as well as \nH3N2 Viruses, have now been cocirculating worldwide for\n29 years and show little evidence of imminent extinction."},"context_chunks":{"kind":"list like","value":["1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.","But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..","Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.","The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.","Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.","But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .","Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.","No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .","These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.","The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .","Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.","Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .","The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.","Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.","Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.","In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.","The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.","The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.","However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?","Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.","And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.","As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.","Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.","Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.","One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.","Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?","One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?","Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.","Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..","The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.","Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.","These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.","The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.","The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .","The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.","But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.","But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.","Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.","In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.","In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.","Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.","Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.","There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.","Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.","Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.","His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.","Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.","Johnson NPAS, Mueller J. 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Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.","A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.","J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.","J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.","Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."],"string":"[\n \"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\\\" and David M. Morens1- The “Spanish\\\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\",\n \"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..\",\n \"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.\",\n \"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.\",\n \"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.\",\n \"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .\",\n \"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.\",\n \"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .\",\n \"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.\",\n \"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .\",\n \"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.\",\n \"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .\",\n \"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.\",\n \"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.\",\n \"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.\",\n \"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.\",\n \"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.\",\n \"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.\",\n \"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?\",\n \"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.\",\n \"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.\",\n \"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.\",\n \"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.\",\n \"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.\",\n \"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.\",\n \"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?\",\n \"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\",\n \"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.\",\n \"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..\",\n \"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.\",\n \"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.\",\n \"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.\",\n \"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.\",\n \"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .\",\n \"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.\",\n \"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.\",\n \"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.\",\n \"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.\",\n \"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.\",\n \"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.\",\n \"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\",\n \"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.\",\n \"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\",\n \"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.\",\n \"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.\",\n \"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.\",\n \"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.\",\n \"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.\",\n \"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.\",\n \"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.\",\n \"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.\",\n \"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.\",\n \"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.\",\n \"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.\",\n \"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.\",\n \"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.\",\n \"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.\",\n \"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.\",\n \"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.\",\n \"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.\"\n]"},"document_id":{"kind":"number","value":2684,"string":"2,684"},"id":{"kind":"number","value":1073,"string":"1,073"}}},{"rowIdx":691,"cells":{"question":{"kind":"string","value":"Is the origin and epidemiology of the 1918 swine flu (Spanish Influenza) known?"},"answer":{"kind":"string","value":"ongoing studies to map Virulence\nfactors are yielding interesting results. The 1918 sequence\ndata, however, leave unanswered questions about the ori-\ngin of the Virus (19) and about the epidemiology of the\npandemic."},"context_chunks":{"kind":"list like","value":["1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.","But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..","Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.","The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.","Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.","But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .","Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.","No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .","These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.","The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .","Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.","Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .","The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.","Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.","Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.","In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.","The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.","The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.","However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?","Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.","And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.","As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.","Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.","Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.","One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.","Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?","One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?","Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.","Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..","The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.","Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.","These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.","The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.","The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .","The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.","But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.","But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.","Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.","In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.","In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.","Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.","Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.","There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.","Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.","Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.","His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. 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Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.","Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."],"string":"[\n \"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\\\" and David M. Morens1- The “Spanish\\\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\",\n \"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..\",\n \"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.\",\n \"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.\",\n \"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.\",\n \"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .\",\n \"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.\",\n \"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .\",\n \"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.\",\n \"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .\",\n \"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.\",\n \"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .\",\n \"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.\",\n \"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.\",\n \"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.\",\n \"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.\",\n \"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.\",\n \"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.\",\n \"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?\",\n \"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.\",\n \"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.\",\n \"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.\",\n \"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.\",\n \"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.\",\n \"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.\",\n \"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?\",\n \"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\",\n \"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.\",\n \"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..\",\n \"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.\",\n \"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.\",\n \"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.\",\n \"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.\",\n \"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .\",\n \"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.\",\n \"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.\",\n \"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.\",\n \"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.\",\n \"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.\",\n \"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.\",\n \"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\",\n \"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.\",\n \"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\",\n \"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.\",\n \"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.\",\n \"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.\",\n \"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.\",\n \"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.\",\n \"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.\",\n \"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.\",\n \"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.\",\n \"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.\",\n \"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.\",\n \"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.\",\n \"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.\",\n \"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.\",\n \"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.\",\n \"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.\",\n \"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.\",\n \"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.\"\n]"},"document_id":{"kind":"number","value":2684,"string":"2,684"},"id":{"kind":"number","value":1075,"string":"1,075"}}},{"rowIdx":692,"cells":{"question":{"kind":"string","value":"What is the geographical origin of the H1N1 swine flu ?"},"answer":{"kind":"string","value":"Historical and epidemiologic data are inade-\nquate to identify the geographic origin of the Virus (21),\nand recent phylogenetic analysis of the 1918 Viral genome\ndoes not place the Virus in any geographic context"},"context_chunks":{"kind":"list like","value":["1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.","But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..","Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.","The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.","Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.","But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .","Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.","No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .","These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.","The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .","Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.","Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .","The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.","Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.","Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.","In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.","The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.","The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.","However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?","Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.","And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.","As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.","Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.","Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.","One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.","Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?","One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?","Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.","Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..","The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.","Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.","These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.","The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.","The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .","The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.","But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.","But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.","Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.","In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.","In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.","Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.","Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.","There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.","Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.","Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.","His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. 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Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.","Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.","Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.","Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.","Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.","Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.","A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.","J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.","J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.","Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."],"string":"[\n \"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\\\" and David M. Morens1- The “Spanish\\\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\",\n \"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..\",\n \"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.\",\n \"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.\",\n \"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.\",\n \"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .\",\n \"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.\",\n \"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .\",\n \"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.\",\n \"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .\",\n \"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.\",\n \"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .\",\n \"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.\",\n \"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.\",\n \"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.\",\n \"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.\",\n \"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.\",\n \"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.\",\n \"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?\",\n \"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.\",\n \"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.\",\n \"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.\",\n \"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.\",\n \"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.\",\n \"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.\",\n \"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?\",\n \"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\",\n \"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.\",\n \"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..\",\n \"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.\",\n \"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.\",\n \"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.\",\n \"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.\",\n \"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .\",\n \"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.\",\n \"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.\",\n \"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.\",\n \"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.\",\n \"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.\",\n \"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.\",\n \"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\",\n \"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.\",\n \"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\",\n \"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.\",\n \"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.\",\n \"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.\",\n \"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.\",\n \"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.\",\n \"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.\",\n \"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.\",\n \"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.\",\n \"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.\",\n \"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.\",\n \"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.\",\n \"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.\",\n \"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.\",\n \"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.\",\n \"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.\",\n \"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.\",\n \"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.\"\n]"},"document_id":{"kind":"number","value":2684,"string":"2,684"},"id":{"kind":"number","value":1082,"string":"1,082"}}},{"rowIdx":693,"cells":{"question":{"kind":"string","value":"Is the geographical origin of the 1918 H1N1 swine flu known?"},"answer":{"kind":"string","value":"Confounding definite assignment of a geographic\npoint of origin, the 1918 pandemic spread more or less\nsimultaneously in 3 distinct waves during an z12-month\nperiod in 191871919, in Europe, Asia, and North America\n(the first wave was best described in the United States in\nMarch 1918)"},"context_chunks":{"kind":"list like","value":["1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.","But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..","Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.","The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.","Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.","But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .","Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.","No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .","These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.","The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .","Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.","Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .","The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.","Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.","Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.","In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.","The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.","The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.","However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?","Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.","And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.","As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.","Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.","Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.","One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.","Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?","One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?","Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.","Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..","The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.","Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.","These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.","The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.","The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .","The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.","But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.","But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.","Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.","In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.","In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.","Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.","Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.","There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.","Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.","Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.","His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.","Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.","Johnson NPAS, Mueller J. 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Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.","12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.","J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.","Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.","Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.","Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.","Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.","Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.","A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.","J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.","J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.","Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."],"string":"[\n \"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\\\" and David M. Morens1- The “Spanish\\\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\",\n \"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..\",\n \"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.\",\n \"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.\",\n \"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.\",\n \"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .\",\n \"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.\",\n \"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .\",\n \"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.\",\n \"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .\",\n \"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.\",\n \"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .\",\n \"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.\",\n \"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.\",\n \"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.\",\n \"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.\",\n \"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.\",\n \"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.\",\n \"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?\",\n \"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.\",\n \"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.\",\n \"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.\",\n \"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.\",\n \"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.\",\n \"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.\",\n \"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?\",\n \"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\",\n \"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.\",\n \"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..\",\n \"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.\",\n \"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.\",\n \"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.\",\n \"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.\",\n \"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .\",\n \"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.\",\n \"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.\",\n \"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.\",\n \"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.\",\n \"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.\",\n \"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.\",\n \"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\",\n \"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.\",\n \"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\",\n \"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.\",\n \"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.\",\n \"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. 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Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.\",\n \"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.\",\n \"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.\",\n \"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.\",\n \"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.\",\n \"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.\",\n \"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.\"\n]"},"document_id":{"kind":"number","value":2684,"string":"2,684"},"id":{"kind":"number","value":1087,"string":"1,087"}}},{"rowIdx":694,"cells":{"question":{"kind":"string","value":"What is an unique feature of the 1918 swine flu?"},"answer":{"kind":"string","value":"the simultaneous (or nearly simultaneous) infection\nof humans and swin"},"context_chunks":{"kind":"list like","value":["1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.","But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..","Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.","The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.","Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.","But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .","Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.","No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .","These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.","The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .","Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.","Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .","The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.","Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.","Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.","In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.","The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.","The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.","However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?","Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.","And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.","As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.","Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.","Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.","One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.","Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?","One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?","Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.","Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..","The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.","Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.","These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.","The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.","The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .","The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.","But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.","But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.","Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.","In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.","In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.","Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.","Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.","There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.","Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.","Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.","His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.","Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.","Johnson NPAS, Mueller J. 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Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.","12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.","J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.","Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.","Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.","Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. 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Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.","A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.","J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.","J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.","Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."],"string":"[\n \"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\\\" and David M. Morens1- The “Spanish\\\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\",\n \"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..\",\n \"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.\",\n \"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.\",\n \"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.\",\n \"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .\",\n \"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.\",\n \"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .\",\n \"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.\",\n \"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .\",\n \"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.\",\n \"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .\",\n \"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.\",\n \"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.\",\n \"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.\",\n \"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.\",\n \"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.\",\n \"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.\",\n \"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?\",\n \"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.\",\n \"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.\",\n \"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.\",\n \"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.\",\n \"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.\",\n \"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.\",\n \"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?\",\n \"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\",\n \"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.\",\n \"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..\",\n \"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.\",\n \"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.\",\n \"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.\",\n \"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.\",\n \"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .\",\n \"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.\",\n \"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.\",\n \"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.\",\n \"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.\",\n \"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.\",\n \"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.\",\n \"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\",\n \"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.\",\n \"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\",\n \"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.\",\n \"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.\",\n \"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.\",\n \"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.\",\n \"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.\",\n \"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.\",\n \"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.\",\n \"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.\",\n \"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.\",\n \"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.\",\n \"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.\",\n \"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.\",\n \"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.\",\n \"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.\",\n \"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.\",\n \"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.\",\n \"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.\"\n]"},"document_id":{"kind":"number","value":2684,"string":"2,684"},"id":{"kind":"number","value":1088,"string":"1,088"}}},{"rowIdx":695,"cells":{"question":{"kind":"string","value":"What season or time of the year do the new strains of influenza emerge?"},"answer":{"kind":"string","value":"Historical records since the 16th century suggest that\nnew influenza pandemics may appear at any time of year,\nnot necessarily in the familiar annual winter patterns of\ninterpandemic years,"},"context_chunks":{"kind":"list like","value":["1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.","But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..","Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.","The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.","Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.","But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .","Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.","No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .","These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.","The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .","Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.","Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .","The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.","Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.","Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.","In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.","The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.","The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.","However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?","Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.","And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.","As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.","Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.","Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.","One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.","Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?","One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?","Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.","Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..","The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.","Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.","These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.","The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.","The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .","The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.","But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.","But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.","Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.","In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.","In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.","Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.","Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.","There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.","Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.","Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.","His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. 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Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.","Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."],"string":"[\n \"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\\\" and David M. Morens1- The “Spanish\\\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\",\n \"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..\",\n \"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.\",\n \"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.\",\n \"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.\",\n \"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .\",\n \"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.\",\n \"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .\",\n \"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.\",\n \"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .\",\n \"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.\",\n \"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .\",\n \"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.\",\n \"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.\",\n \"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.\",\n \"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.\",\n \"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.\",\n \"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.\",\n \"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?\",\n \"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.\",\n \"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.\",\n \"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.\",\n \"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.\",\n \"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.\",\n \"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.\",\n \"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?\",\n \"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\",\n \"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.\",\n \"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..\",\n \"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.\",\n \"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.\",\n \"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.\",\n \"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.\",\n \"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .\",\n \"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.\",\n \"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.\",\n \"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.\",\n \"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.\",\n \"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.\",\n \"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.\",\n \"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\",\n \"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.\",\n \"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\",\n \"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.\",\n \"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.\",\n \"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.\",\n \"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.\",\n \"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.\",\n \"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.\",\n \"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.\",\n \"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.\",\n \"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.\",\n \"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.\",\n \"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.\",\n \"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.\",\n \"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.\",\n \"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.\",\n \"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.\",\n \"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.\",\n \"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.\"\n]"},"document_id":{"kind":"number","value":2684,"string":"2,684"},"id":{"kind":"number","value":1089,"string":"1,089"}}},{"rowIdx":696,"cells":{"question":{"kind":"string","value":"Once appeared, when do the influenza like diseases occur in subsequent years?"},"answer":{"kind":"string","value":"confronted by the selection pressures of population immu-\nnity, these pandemic Viruses begin to drift genetically and\neventually settle into a pattern of annual epidemic recur-\nrences caused by the drifted Virus variants."},"context_chunks":{"kind":"list like","value":["1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.","But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..","Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.","The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.","Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.","But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .","Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.","No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .","These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.","The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .","Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.","Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .","The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.","Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.","Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.","In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.","The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.","The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.","However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?","Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.","And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.","As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.","Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.","Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.","One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.","Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?","One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?","Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.","Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..","The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.","Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.","These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.","The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.","The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .","The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.","But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.","But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.","Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.","In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.","In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.","Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.","Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.","There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.","Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.","Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.","His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. 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Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.","A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.","J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.","J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.","Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."],"string":"[\n \"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\\\" and David M. Morens1- The “Spanish\\\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\",\n \"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..\",\n \"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.\",\n \"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.\",\n \"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.\",\n \"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .\",\n \"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.\",\n \"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .\",\n \"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.\",\n \"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .\",\n \"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.\",\n \"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .\",\n \"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.\",\n \"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.\",\n \"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.\",\n \"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.\",\n \"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.\",\n \"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.\",\n \"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?\",\n \"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.\",\n \"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.\",\n \"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.\",\n \"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.\",\n \"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.\",\n \"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.\",\n \"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?\",\n \"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\",\n \"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.\",\n \"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..\",\n \"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.\",\n \"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.\",\n \"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.\",\n \"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.\",\n \"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .\",\n \"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.\",\n \"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.\",\n \"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.\",\n \"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.\",\n \"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.\",\n \"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.\",\n \"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\",\n \"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.\",\n \"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\",\n \"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.\",\n \"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.\",\n \"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.\",\n \"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.\",\n \"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.\",\n \"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.\",\n \"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.\",\n \"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.\",\n \"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.\",\n \"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.\",\n \"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.\",\n \"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.\",\n \"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.\",\n \"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.\",\n \"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.\",\n \"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.\",\n \"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.\"\n]"},"document_id":{"kind":"number","value":2684,"string":"2,684"},"id":{"kind":"number","value":1090,"string":"1,090"}}},{"rowIdx":697,"cells":{"question":{"kind":"string","value":"When did the first wave of the H1N1 swine flu (Spanish Influenza) occur?"},"answer":{"kind":"string","value":" a first or spring wave\nbegan in March 1918 and spread unevenly through the\nUnited States, Europe, and possibly Asia over the next 6\nmonths"},"context_chunks":{"kind":"list like","value":["1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.","But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..","Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.","The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.","Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.","But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .","Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.","No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .","These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.","The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .","Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.","Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .","The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.","Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.","Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.","In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.","The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.","The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.","However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?","Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.","And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.","As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.","Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.","Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.","One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.","Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?","One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?","Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.","Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..","The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.","Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.","These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.","The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.","The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .","The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.","But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.","But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.","Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.","In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.","In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.","Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.","Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.","There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.","Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.","Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.","His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.","Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.","Johnson NPAS, Mueller J. 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Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.","12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.","J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.","Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.","Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.","Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.","Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.","Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.","A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.","J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.","J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.","Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."],"string":"[\n \"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\\\" and David M. Morens1- The “Spanish\\\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\",\n \"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..\",\n \"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.\",\n \"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.\",\n \"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.\",\n \"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .\",\n \"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.\",\n \"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .\",\n \"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.\",\n \"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .\",\n \"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.\",\n \"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .\",\n \"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.\",\n \"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.\",\n \"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.\",\n \"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.\",\n \"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.\",\n \"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.\",\n \"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?\",\n \"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.\",\n \"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.\",\n \"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.\",\n \"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.\",\n \"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.\",\n \"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.\",\n \"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?\",\n \"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\",\n \"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.\",\n \"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..\",\n \"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.\",\n \"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.\",\n \"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.\",\n \"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.\",\n \"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .\",\n \"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.\",\n \"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.\",\n \"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.\",\n \"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.\",\n \"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.\",\n \"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.\",\n \"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\",\n \"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.\",\n \"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\",\n \"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.\",\n \"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.\",\n \"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.\",\n \"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.\",\n \"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.\",\n \"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.\",\n \"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.\",\n \"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.\",\n \"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.\",\n \"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.\",\n \"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.\",\n \"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.\",\n \"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.\",\n \"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.\",\n \"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.\",\n \"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.\",\n \"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.\"\n]"},"document_id":{"kind":"number","value":2684,"string":"2,684"},"id":{"kind":"number","value":1091,"string":"1,091"}}},{"rowIdx":698,"cells":{"question":{"kind":"string","value":"What was the death rate in the first wave of the 1918 swine flu pandemic?"},"answer":{"kind":"string","value":" Illness rates were high, but death rates\nin most locales were not appreciably above normal."},"context_chunks":{"kind":"list like","value":["1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.","But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..","Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.","The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.","Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.","But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .","Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.","No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .","These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.","The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .","Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.","Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .","The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.","Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.","Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.","In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.","The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.","The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.","However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?","Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.","And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.","As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.","Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.","Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.","One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.","Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?","One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?","Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.","Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..","The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.","Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.","These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.","The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.","The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .","The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.","But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.","But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.","Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.","In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.","In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.","Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.","Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.","There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.","Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.","Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.","His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.","Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.","Johnson NPAS, Mueller J. 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Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.","12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.","J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.","Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.","Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.","Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.","Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.","Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.","A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.","J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.","J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.","Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."],"string":"[\n \"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\\\" and David M. Morens1- The “Spanish\\\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\",\n \"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..\",\n \"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.\",\n \"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.\",\n \"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.\",\n \"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .\",\n \"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.\",\n \"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .\",\n \"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.\",\n \"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .\",\n \"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.\",\n \"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .\",\n \"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.\",\n \"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.\",\n \"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.\",\n \"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.\",\n \"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.\",\n \"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.\",\n \"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?\",\n \"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.\",\n \"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.\",\n \"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.\",\n \"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.\",\n \"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.\",\n \"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.\",\n \"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?\",\n \"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\",\n \"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.\",\n \"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..\",\n \"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.\",\n \"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.\",\n \"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.\",\n \"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.\",\n \"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .\",\n \"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.\",\n \"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.\",\n \"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.\",\n \"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.\",\n \"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.\",\n \"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.\",\n \"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\",\n \"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.\",\n \"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\",\n \"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.\",\n \"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.\",\n \"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. 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Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.\",\n \"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.\",\n \"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.\",\n \"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.\",\n \"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.\",\n \"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.\",\n \"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.\",\n \"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.\",\n \"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.\"\n]"},"document_id":{"kind":"number","value":2684,"string":"2,684"},"id":{"kind":"number","value":1092,"string":"1,092"}}},{"rowIdx":699,"cells":{"question":{"kind":"string","value":"When were the second and the third wave of the 1918-1919 swine flu pandemic?"},"answer":{"kind":"string","value":" A sec-\nond or fall wave spread globally from September to\nNovember 1918 and was highly fatal. In many nations, a\nthird wave occurred in early 1919 "},"context_chunks":{"kind":"list like","value":["1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.","But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..","Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.","The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.","Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.","But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .","Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.","No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .","These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.","The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .","Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.","Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .","The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.","Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.","Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.","In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.","The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.","The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.","However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?","Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.","And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.","As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.","Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.","Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.","One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.","Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?","One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?","Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.","Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..","The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.","Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.","These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.","The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.","The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .","The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.","But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.","But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.","Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.","In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.","In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.","Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.","Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.","There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.","Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.","Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.","His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.","Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.","Johnson NPAS, Mueller J. 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Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.","12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.","J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.","Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.","Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.","Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. 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Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.","A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.","J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.","J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.","Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."],"string":"[\n \"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\\\" and David M. Morens1- The “Spanish\\\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\",\n \"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..\",\n \"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.\",\n \"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.\",\n \"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.\",\n \"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .\",\n \"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.\",\n \"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .\",\n \"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.\",\n \"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .\",\n \"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.\",\n \"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .\",\n \"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.\",\n \"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.\",\n \"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.\",\n \"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.\",\n \"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.\",\n \"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.\",\n \"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?\",\n \"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.\",\n \"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.\",\n \"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.\",\n \"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.\",\n \"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.\",\n \"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.\",\n \"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?\",\n \"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\",\n \"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.\",\n \"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..\",\n \"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.\",\n \"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.\",\n \"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.\",\n \"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.\",\n \"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .\",\n \"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.\",\n \"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.\",\n \"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.\",\n \"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.\",\n \"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.\",\n \"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.\",\n \"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\",\n \"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.\",\n \"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\",\n \"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.\",\n \"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.\",\n \"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.\",\n \"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.\",\n \"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.\",\n \"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.\",\n \"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.\",\n \"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.\",\n \"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.\",\n \"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.\",\n \"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.\",\n \"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.\",\n \"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.\",\n \"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.\",\n \"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.\",\n \"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.\",\n \"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.\"\n]"},"document_id":{"kind":"number","value":2684,"string":"2,684"},"id":{"kind":"number","value":1093,"string":"1,093"}}}],"truncated":false,"partial":false},"paginationData":{"pageIndex":6,"numItemsPerPage":100,"numTotalItems":814,"offset":600,"length":100}},"jwt":"eyJhbGciOiJFZERTQSJ9.eyJyZWFkIjp0cnVlLCJwZXJtaXNzaW9ucyI6eyJyZXBvLmNvbnRlbnQucmVhZCI6dHJ1ZX0sImlhdCI6MTcyODk5ODY4MCwic3ViIjoiL2RhdGFzZXRzL21pbmgyMS9DT1ZJRC1RQS1xdWVzdGlvbi1hbnN3ZXJpbmctYmllbmNvZGVyLWRhdGEtNDVfNDVfMTAiLCJleHAiOjE3MjkwMDIyODAsImlzcyI6Imh0dHBzOi8vaHVnZ2luZ2ZhY2UuY28ifQ.8ymAZPIflIAVZNtS09dhujRQpqH89pnwUXMFAHevn6vwA27WxgaHK3mKmUG0vLEV2KZdtqgkDCIDCLuMuS9cCg","displayUrls":true},"discussionsStats":{"closed":0,"open":0,"total":0},"fullWidth":true,"hasGatedAccess":true,"hasFullAccess":true,"isEmbedded":false}">
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What is NPA? | nasopharyngeal aspirates | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What is BAL? | bronchoalveolar lavage | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,268 |
What is the the proportion of deaths among those infected with MERS-CoV? | is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What is the the proportion of deaths among those infected with MERS-CoV? | above that for SARS-CoV | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What has prevented worldwide spread of MERS-COV? | the low transmission rate | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What must be reported to the world organization for animal health as an emerging disease ? | animal MERS-CoV infections | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What are juvenile DCs more often positive for? | virus or viral RNA | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,277 |
What are older DCs are more likely to be positive for? | seropositive and RNA or virus negative | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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Why is there an increased risk to humans of spill-over during calving season? | due to new infections among naïve DC populations | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,280 |
Which may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV ? | slaughterhouse workers who kill both younger and older DCs | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,281 |
How long after Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered? | up to 48 h | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,282 |
How long after Infectious MERS-CoV added to DC, goat or cow milk and stored at 22°C could be recovered? | up to 48 h | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What ablated MERS-COV infection completely? | pasteurization | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,284 |
How long MERS-CoV remained viable at high ambient temperature (30°C) and low RH (30 %) ? | for 24 h | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What is considered the mechanism of human-to-human transmission of MERS-COV? | Droplet spread between humans | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What is the transmission of MERS-CoV is defined as? | sporadic | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What is the transmission of MERS-CoV is defined as? | intra-familial, | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What is the transmission of MERS-CoV is defined as? | often healthcare associated | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What is the transmission of MERS-CoV is defined as? | inefficient | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What is the transmission of MERS-CoV is defined as? | requiring close and prolonged contact | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What is the the rate of general transmission, even in outbreaks? | readily transmit to more than one other human | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What do the majority of human cases of MERS-CoV seem not to do? | readily transmit to more than one other human | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,296 |
What is the basic reproduction number (R 0)? | the average number of infections caused by one infected individual in a fully susceptible population | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,298 |
What is the basic reproduction number (R 0 ) for MERS-COV? | close to one | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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Why has MERS had a constant presence in the Arabian Peninsula? | due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks.
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"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,300 |
What was the first known MERS human-to-human transmission event was one characterized by? | acute LRT disease | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,301 |
Where was the first known MERS human-to-human transmission event? | in a healthcare setting in Jordan | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,302 |
Approximately what percentage of MERS cases were fatal in KSA? | 21 % | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,367 |
Approximately what percentage of MERS cases were died outside KSA? | 21 % | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,368 |
What percentage of HCWs comprised of MERS cases in the KSA and South Korea? | 16 % | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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How has most of the analysis of MERS-CoV genetics been performed? | using high throughput or "deep" sequencing methods for complete genome deduction | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,370 |
What does Clade A contain? | only human-derived MERS-CoV genomes | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,372 |
What does clade B comprise? | the majority of human and camel genomes deduced thus far | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,373 |
How many clades have become apparent in genome of MERS-COV from humans and DCs? | has had more MERS cases than any other region of the KSA | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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Which city has had has had more MERS cases than any other region of the KSA ? | Riyadh | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,374 |
Which city harbours a wide range of MERS-CoV variants | Riyadh | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,375 |
How has the vast majority of MERS-CoV transmission occurred? | from infected to uninfected humans in close and prolonged contact | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,376 |
How were the transmission circumstances created? | by poor infection control in health care settings | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,377 |
What percentage of humans have died among all humans reported to be infected? | nearly 40 % | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What would aid accurate calculation of a case fatality ratio? | Global alignment of case definitions | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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How does MARS-COV differ from SARS-COV? | MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.
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"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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Is there any evidence that MERS-CoV is a virus of pandemic concern? | There is no evidence that MERS-CoV is a virus of pandemic concern | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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How did the first WHO case definition define probable cases of MERS? | based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement. | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,215 |
What illness is caused by the 2019-nCOV Coronavirus? | The novel coronavirus (2019-nCoV) infection caused pneumonia. | [
"The novel coronavirus 2019-nCoV infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood 6 of 57 patients and the anal swabs 11 of 28 patients . Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity p-value = 0.0001 . Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab Ct value = 24 + 39 was higher than in the blood Ct value = 34 + 39 from patient 2, suggesting that the virus might replicate in the digestive tract.",
"Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab Ct value = 24 + 39 was higher than in the blood Ct value = 34 + 39 from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus 2019-nCoV , originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination , transmission and evolution , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence.",
"Basic scientific research has achieved significantly in the investigation of viral origination , transmission and evolution , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital.",
"Several studies have roughly described the overall clinical features of 2019-nCoV patients . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes.",
"In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit Da'an Gene Corporation, Cat: DA0630 on an automatic workstation Smart 32 Da'an Gene Corporation following the guidelines. Real-time reverse transcriptional polymerase chain reaction RT-PCR reagent Da'an Gene cooperation, Cat DA0930 was employed for viral detection per the protocol.",
"Viral RNA was extracted with Nucleic Acid Isolation Kit Da'an Gene Corporation, Cat: DA0630 on an automatic workstation Smart 32 Da'an Gene Corporation following the guidelines. Real-time reverse transcriptional polymerase chain reaction RT-PCR reagent Da'an Gene cooperation, Cat DA0930 was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab FAM reporter and N VIC reporter genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set s gave a reliable signal s . All patients had pneumonia-based diseases but with diversified clinical manifestation.",
"Samples were considered to be viral positive when either or both set s gave a reliable signal s . All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1 distress of respiratory with respiratory rate > = 30/min; 2 Oxygen saturation < = 93% in the rest state, and 3 arterial oxygen tension PaO₂ over inspiratory oxygen fraction FIO₂ of less than 300 mm Hg. In the blood detection cohort Figure 1 A , patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them 100% were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them 23.5% were severe cases.",
"In the blood detection cohort Figure 1 A , patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them 100% were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them 23.5% were severe cases. The ratio of severe symptoms between these two groups was significantly different p value = 0.0001 . In the anal swab cohort Figure 1 B , 11 of 28 cases were detected to be anal swab positive, 8 of them 72.7% were with severe symptoms, which was significantly higher than that 4 23.5% of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 Figure 2 A was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing.",
"Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 Figure 2 A was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 Ct = 17 + 25 , 7, 8 Ct = 25 + 26 , and 11 Ct = 15 + 25 . In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal Ct = Neg+39 , and then the signal was gone again on day 8. On day 9, a low level of viral RNA Ct = 36 + 41 was detected again in the blood.",
"In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal Ct = Neg+39 , and then the signal was gone again on day 8. On day 9, a low level of viral RNA Ct = 36 + 41 was detected again in the blood. On day 12, the blood lost signal again. A high concentration of virus RNA Ct = 23 + 27 was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 Figure 2 B , who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes.",
"Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 Figure 2 B , who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 Ct = 34 + 36 and 8 Ct = 38 + 38 . His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus Ct = 25 + 36 were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal Ct = 23 + 24 , blood Ct = 34 + 39 and anal Ct = 24 + 29 samples on day 10.",
"Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus Ct = 25 + 36 were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal Ct = 23 + 24 , blood Ct = 34 + 39 and anal Ct = 24 + 29 samples on day 10. He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 Figure 3 A was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal Ct = 30 + 30 and blood samples Ct = 37 + 39 on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22.",
"Patient 3 Figure 3 A was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal Ct = 30 + 30 and blood samples Ct = 37 + 39 on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 Figure 3 B was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive Ct = 32 + 37 . On day 9, he was transferred out. Patient 5 Figure 3 C was admitted on illness day 4 and his blood sample was virus-positive Ct = 38 + Neg on day 6.",
"On day 9, he was transferred out. Patient 5 Figure 3 C was admitted on illness day 4 and his blood sample was virus-positive Ct = 38 + Neg on day 6. Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 Figure 3 D with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory.",
"As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in or later progressed to severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group p = 0.0001 . Patient 2 Figure 2 B , 5 Figure 3 C and 6 Figure 3 D all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 Figure 2 A and 3 Figure 3 A who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 Figure 3 B who had serum sample collected one day post the diagnosis of severe illness.",
"Patient 2 Figure 2 B , 5 Figure 3 C and 6 Figure 3 D all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 Figure 2 A and 3 Figure 3 A who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 Figure 3 B who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity p = 0.0102 . The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region.",
"The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA Ct = 23 + 27, on day 13 was detected in anal swab but not in pharyngeal the same day and blood 1 d ahead . For patient 2, higher concentrations of viral RNAs were detected in anal swab Ct = 24 + 39 and pharyngeal swab Ct = 23 + 24 than in the blood Ct = 34 + 39 on the same day.",
"For patient 1, a high concentration of viral RNA Ct = 23 + 27, on day 13 was detected in anal swab but not in pharyngeal the same day and blood 1 d ahead . For patient 2, higher concentrations of viral RNAs were detected in anal swab Ct = 24 + 39 and pharyngeal swab Ct = 23 + 24 than in the blood Ct = 34 + 39 on the same day. Angiotensin-converting enzyme 2 ACE2 still is one of the receptors for 2019-nCoV attachment and entry . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body.",
"Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool.",
"Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction."
] | 2,519 | 1,149 |
In addition to oral swabs, which tests detected the presence of 2019-nCOV virus? | the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). | [
"The novel coronavirus 2019-nCoV infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood 6 of 57 patients and the anal swabs 11 of 28 patients . Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity p-value = 0.0001 . Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab Ct value = 24 + 39 was higher than in the blood Ct value = 34 + 39 from patient 2, suggesting that the virus might replicate in the digestive tract.",
"Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab Ct value = 24 + 39 was higher than in the blood Ct value = 34 + 39 from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus 2019-nCoV , originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination , transmission and evolution , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence.",
"Basic scientific research has achieved significantly in the investigation of viral origination , transmission and evolution , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital.",
"Several studies have roughly described the overall clinical features of 2019-nCoV patients . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes.",
"In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit Da'an Gene Corporation, Cat: DA0630 on an automatic workstation Smart 32 Da'an Gene Corporation following the guidelines. Real-time reverse transcriptional polymerase chain reaction RT-PCR reagent Da'an Gene cooperation, Cat DA0930 was employed for viral detection per the protocol.",
"Viral RNA was extracted with Nucleic Acid Isolation Kit Da'an Gene Corporation, Cat: DA0630 on an automatic workstation Smart 32 Da'an Gene Corporation following the guidelines. Real-time reverse transcriptional polymerase chain reaction RT-PCR reagent Da'an Gene cooperation, Cat DA0930 was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab FAM reporter and N VIC reporter genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set s gave a reliable signal s . All patients had pneumonia-based diseases but with diversified clinical manifestation.",
"Samples were considered to be viral positive when either or both set s gave a reliable signal s . All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1 distress of respiratory with respiratory rate > = 30/min; 2 Oxygen saturation < = 93% in the rest state, and 3 arterial oxygen tension PaO₂ over inspiratory oxygen fraction FIO₂ of less than 300 mm Hg. In the blood detection cohort Figure 1 A , patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them 100% were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them 23.5% were severe cases.",
"In the blood detection cohort Figure 1 A , patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them 100% were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them 23.5% were severe cases. The ratio of severe symptoms between these two groups was significantly different p value = 0.0001 . In the anal swab cohort Figure 1 B , 11 of 28 cases were detected to be anal swab positive, 8 of them 72.7% were with severe symptoms, which was significantly higher than that 4 23.5% of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 Figure 2 A was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing.",
"Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 Figure 2 A was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 Ct = 17 + 25 , 7, 8 Ct = 25 + 26 , and 11 Ct = 15 + 25 . In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal Ct = Neg+39 , and then the signal was gone again on day 8. On day 9, a low level of viral RNA Ct = 36 + 41 was detected again in the blood.",
"In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal Ct = Neg+39 , and then the signal was gone again on day 8. On day 9, a low level of viral RNA Ct = 36 + 41 was detected again in the blood. On day 12, the blood lost signal again. A high concentration of virus RNA Ct = 23 + 27 was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 Figure 2 B , who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes.",
"Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 Figure 2 B , who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 Ct = 34 + 36 and 8 Ct = 38 + 38 . His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus Ct = 25 + 36 were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal Ct = 23 + 24 , blood Ct = 34 + 39 and anal Ct = 24 + 29 samples on day 10.",
"Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus Ct = 25 + 36 were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal Ct = 23 + 24 , blood Ct = 34 + 39 and anal Ct = 24 + 29 samples on day 10. He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 Figure 3 A was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal Ct = 30 + 30 and blood samples Ct = 37 + 39 on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22.",
"Patient 3 Figure 3 A was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal Ct = 30 + 30 and blood samples Ct = 37 + 39 on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 Figure 3 B was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive Ct = 32 + 37 . On day 9, he was transferred out. Patient 5 Figure 3 C was admitted on illness day 4 and his blood sample was virus-positive Ct = 38 + Neg on day 6.",
"On day 9, he was transferred out. Patient 5 Figure 3 C was admitted on illness day 4 and his blood sample was virus-positive Ct = 38 + Neg on day 6. Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 Figure 3 D with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory.",
"As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in or later progressed to severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group p = 0.0001 . Patient 2 Figure 2 B , 5 Figure 3 C and 6 Figure 3 D all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 Figure 2 A and 3 Figure 3 A who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 Figure 3 B who had serum sample collected one day post the diagnosis of severe illness.",
"Patient 2 Figure 2 B , 5 Figure 3 C and 6 Figure 3 D all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 Figure 2 A and 3 Figure 3 A who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 Figure 3 B who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity p = 0.0102 . The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region.",
"The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA Ct = 23 + 27, on day 13 was detected in anal swab but not in pharyngeal the same day and blood 1 d ahead . For patient 2, higher concentrations of viral RNAs were detected in anal swab Ct = 24 + 39 and pharyngeal swab Ct = 23 + 24 than in the blood Ct = 34 + 39 on the same day.",
"For patient 1, a high concentration of viral RNA Ct = 23 + 27, on day 13 was detected in anal swab but not in pharyngeal the same day and blood 1 d ahead . For patient 2, higher concentrations of viral RNAs were detected in anal swab Ct = 24 + 39 and pharyngeal swab Ct = 23 + 24 than in the blood Ct = 34 + 39 on the same day. Angiotensin-converting enzyme 2 ACE2 still is one of the receptors for 2019-nCoV attachment and entry . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body.",
"Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool.",
"Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction."
] | 2,519 | 1,151 |
What is the relationship between the presence of virus in blood and anal swabs and disease severity? | all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. | [
"The novel coronavirus 2019-nCoV infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood 6 of 57 patients and the anal swabs 11 of 28 patients . Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity p-value = 0.0001 . Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab Ct value = 24 + 39 was higher than in the blood Ct value = 34 + 39 from patient 2, suggesting that the virus might replicate in the digestive tract.",
"Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab Ct value = 24 + 39 was higher than in the blood Ct value = 34 + 39 from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus 2019-nCoV , originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination , transmission and evolution , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence.",
"Basic scientific research has achieved significantly in the investigation of viral origination , transmission and evolution , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital.",
"Several studies have roughly described the overall clinical features of 2019-nCoV patients . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes.",
"In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit Da'an Gene Corporation, Cat: DA0630 on an automatic workstation Smart 32 Da'an Gene Corporation following the guidelines. Real-time reverse transcriptional polymerase chain reaction RT-PCR reagent Da'an Gene cooperation, Cat DA0930 was employed for viral detection per the protocol.",
"Viral RNA was extracted with Nucleic Acid Isolation Kit Da'an Gene Corporation, Cat: DA0630 on an automatic workstation Smart 32 Da'an Gene Corporation following the guidelines. Real-time reverse transcriptional polymerase chain reaction RT-PCR reagent Da'an Gene cooperation, Cat DA0930 was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab FAM reporter and N VIC reporter genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set s gave a reliable signal s . All patients had pneumonia-based diseases but with diversified clinical manifestation.",
"Samples were considered to be viral positive when either or both set s gave a reliable signal s . All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1 distress of respiratory with respiratory rate > = 30/min; 2 Oxygen saturation < = 93% in the rest state, and 3 arterial oxygen tension PaO₂ over inspiratory oxygen fraction FIO₂ of less than 300 mm Hg. In the blood detection cohort Figure 1 A , patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them 100% were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them 23.5% were severe cases.",
"In the blood detection cohort Figure 1 A , patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them 100% were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them 23.5% were severe cases. The ratio of severe symptoms between these two groups was significantly different p value = 0.0001 . In the anal swab cohort Figure 1 B , 11 of 28 cases were detected to be anal swab positive, 8 of them 72.7% were with severe symptoms, which was significantly higher than that 4 23.5% of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 Figure 2 A was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing.",
"Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 Figure 2 A was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 Ct = 17 + 25 , 7, 8 Ct = 25 + 26 , and 11 Ct = 15 + 25 . In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal Ct = Neg+39 , and then the signal was gone again on day 8. On day 9, a low level of viral RNA Ct = 36 + 41 was detected again in the blood.",
"In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal Ct = Neg+39 , and then the signal was gone again on day 8. On day 9, a low level of viral RNA Ct = 36 + 41 was detected again in the blood. On day 12, the blood lost signal again. A high concentration of virus RNA Ct = 23 + 27 was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 Figure 2 B , who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes.",
"Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 Figure 2 B , who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 Ct = 34 + 36 and 8 Ct = 38 + 38 . His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus Ct = 25 + 36 were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal Ct = 23 + 24 , blood Ct = 34 + 39 and anal Ct = 24 + 29 samples on day 10.",
"Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus Ct = 25 + 36 were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal Ct = 23 + 24 , blood Ct = 34 + 39 and anal Ct = 24 + 29 samples on day 10. He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 Figure 3 A was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal Ct = 30 + 30 and blood samples Ct = 37 + 39 on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22.",
"Patient 3 Figure 3 A was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal Ct = 30 + 30 and blood samples Ct = 37 + 39 on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 Figure 3 B was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive Ct = 32 + 37 . On day 9, he was transferred out. Patient 5 Figure 3 C was admitted on illness day 4 and his blood sample was virus-positive Ct = 38 + Neg on day 6.",
"On day 9, he was transferred out. Patient 5 Figure 3 C was admitted on illness day 4 and his blood sample was virus-positive Ct = 38 + Neg on day 6. Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 Figure 3 D with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory.",
"As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in or later progressed to severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group p = 0.0001 . Patient 2 Figure 2 B , 5 Figure 3 C and 6 Figure 3 D all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 Figure 2 A and 3 Figure 3 A who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 Figure 3 B who had serum sample collected one day post the diagnosis of severe illness.",
"Patient 2 Figure 2 B , 5 Figure 3 C and 6 Figure 3 D all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 Figure 2 A and 3 Figure 3 A who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 Figure 3 B who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity p = 0.0102 . The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region.",
"The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA Ct = 23 + 27, on day 13 was detected in anal swab but not in pharyngeal the same day and blood 1 d ahead . For patient 2, higher concentrations of viral RNAs were detected in anal swab Ct = 24 + 39 and pharyngeal swab Ct = 23 + 24 than in the blood Ct = 34 + 39 on the same day.",
"For patient 1, a high concentration of viral RNA Ct = 23 + 27, on day 13 was detected in anal swab but not in pharyngeal the same day and blood 1 d ahead . For patient 2, higher concentrations of viral RNAs were detected in anal swab Ct = 24 + 39 and pharyngeal swab Ct = 23 + 24 than in the blood Ct = 34 + 39 on the same day. Angiotensin-converting enzyme 2 ACE2 still is one of the receptors for 2019-nCoV attachment and entry . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body.",
"Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool.",
"Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction."
] | 2,519 | 1,160 |
Which patients were classified as severe in Chinese guidelines? | Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included. | [
"The novel coronavirus 2019-nCoV infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood 6 of 57 patients and the anal swabs 11 of 28 patients . Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity p-value = 0.0001 . Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab Ct value = 24 + 39 was higher than in the blood Ct value = 34 + 39 from patient 2, suggesting that the virus might replicate in the digestive tract.",
"Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab Ct value = 24 + 39 was higher than in the blood Ct value = 34 + 39 from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus 2019-nCoV , originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination , transmission and evolution , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence.",
"Basic scientific research has achieved significantly in the investigation of viral origination , transmission and evolution , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital.",
"Several studies have roughly described the overall clinical features of 2019-nCoV patients . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes.",
"In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit Da'an Gene Corporation, Cat: DA0630 on an automatic workstation Smart 32 Da'an Gene Corporation following the guidelines. Real-time reverse transcriptional polymerase chain reaction RT-PCR reagent Da'an Gene cooperation, Cat DA0930 was employed for viral detection per the protocol.",
"Viral RNA was extracted with Nucleic Acid Isolation Kit Da'an Gene Corporation, Cat: DA0630 on an automatic workstation Smart 32 Da'an Gene Corporation following the guidelines. Real-time reverse transcriptional polymerase chain reaction RT-PCR reagent Da'an Gene cooperation, Cat DA0930 was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab FAM reporter and N VIC reporter genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set s gave a reliable signal s . All patients had pneumonia-based diseases but with diversified clinical manifestation.",
"Samples were considered to be viral positive when either or both set s gave a reliable signal s . All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1 distress of respiratory with respiratory rate > = 30/min; 2 Oxygen saturation < = 93% in the rest state, and 3 arterial oxygen tension PaO₂ over inspiratory oxygen fraction FIO₂ of less than 300 mm Hg. In the blood detection cohort Figure 1 A , patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them 100% were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them 23.5% were severe cases.",
"In the blood detection cohort Figure 1 A , patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them 100% were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them 23.5% were severe cases. The ratio of severe symptoms between these two groups was significantly different p value = 0.0001 . In the anal swab cohort Figure 1 B , 11 of 28 cases were detected to be anal swab positive, 8 of them 72.7% were with severe symptoms, which was significantly higher than that 4 23.5% of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 Figure 2 A was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing.",
"Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 Figure 2 A was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 Ct = 17 + 25 , 7, 8 Ct = 25 + 26 , and 11 Ct = 15 + 25 . In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal Ct = Neg+39 , and then the signal was gone again on day 8. On day 9, a low level of viral RNA Ct = 36 + 41 was detected again in the blood.",
"In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal Ct = Neg+39 , and then the signal was gone again on day 8. On day 9, a low level of viral RNA Ct = 36 + 41 was detected again in the blood. On day 12, the blood lost signal again. A high concentration of virus RNA Ct = 23 + 27 was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 Figure 2 B , who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes.",
"Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 Figure 2 B , who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 Ct = 34 + 36 and 8 Ct = 38 + 38 . His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus Ct = 25 + 36 were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal Ct = 23 + 24 , blood Ct = 34 + 39 and anal Ct = 24 + 29 samples on day 10.",
"Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus Ct = 25 + 36 were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal Ct = 23 + 24 , blood Ct = 34 + 39 and anal Ct = 24 + 29 samples on day 10. He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 Figure 3 A was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal Ct = 30 + 30 and blood samples Ct = 37 + 39 on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22.",
"Patient 3 Figure 3 A was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal Ct = 30 + 30 and blood samples Ct = 37 + 39 on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 Figure 3 B was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive Ct = 32 + 37 . On day 9, he was transferred out. Patient 5 Figure 3 C was admitted on illness day 4 and his blood sample was virus-positive Ct = 38 + Neg on day 6.",
"On day 9, he was transferred out. Patient 5 Figure 3 C was admitted on illness day 4 and his blood sample was virus-positive Ct = 38 + Neg on day 6. Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 Figure 3 D with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory.",
"As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in or later progressed to severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group p = 0.0001 . Patient 2 Figure 2 B , 5 Figure 3 C and 6 Figure 3 D all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 Figure 2 A and 3 Figure 3 A who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 Figure 3 B who had serum sample collected one day post the diagnosis of severe illness.",
"Patient 2 Figure 2 B , 5 Figure 3 C and 6 Figure 3 D all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 Figure 2 A and 3 Figure 3 A who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 Figure 3 B who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity p = 0.0102 . The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region.",
"The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA Ct = 23 + 27, on day 13 was detected in anal swab but not in pharyngeal the same day and blood 1 d ahead . For patient 2, higher concentrations of viral RNAs were detected in anal swab Ct = 24 + 39 and pharyngeal swab Ct = 23 + 24 than in the blood Ct = 34 + 39 on the same day.",
"For patient 1, a high concentration of viral RNA Ct = 23 + 27, on day 13 was detected in anal swab but not in pharyngeal the same day and blood 1 d ahead . For patient 2, higher concentrations of viral RNAs were detected in anal swab Ct = 24 + 39 and pharyngeal swab Ct = 23 + 24 than in the blood Ct = 34 + 39 on the same day. Angiotensin-converting enzyme 2 ACE2 still is one of the receptors for 2019-nCoV attachment and entry . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body.",
"Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool.",
"Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction."
] | 2,519 | 1,168 |
What is the relationship between the presence of virus in blood sample and disease severity? | In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases. | [
"The novel coronavirus 2019-nCoV infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood 6 of 57 patients and the anal swabs 11 of 28 patients . Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity p-value = 0.0001 . Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab Ct value = 24 + 39 was higher than in the blood Ct value = 34 + 39 from patient 2, suggesting that the virus might replicate in the digestive tract.",
"Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab Ct value = 24 + 39 was higher than in the blood Ct value = 34 + 39 from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus 2019-nCoV , originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination , transmission and evolution , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence.",
"Basic scientific research has achieved significantly in the investigation of viral origination , transmission and evolution , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital.",
"Several studies have roughly described the overall clinical features of 2019-nCoV patients . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes.",
"In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit Da'an Gene Corporation, Cat: DA0630 on an automatic workstation Smart 32 Da'an Gene Corporation following the guidelines. Real-time reverse transcriptional polymerase chain reaction RT-PCR reagent Da'an Gene cooperation, Cat DA0930 was employed for viral detection per the protocol.",
"Viral RNA was extracted with Nucleic Acid Isolation Kit Da'an Gene Corporation, Cat: DA0630 on an automatic workstation Smart 32 Da'an Gene Corporation following the guidelines. Real-time reverse transcriptional polymerase chain reaction RT-PCR reagent Da'an Gene cooperation, Cat DA0930 was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab FAM reporter and N VIC reporter genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set s gave a reliable signal s . All patients had pneumonia-based diseases but with diversified clinical manifestation.",
"Samples were considered to be viral positive when either or both set s gave a reliable signal s . All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1 distress of respiratory with respiratory rate > = 30/min; 2 Oxygen saturation < = 93% in the rest state, and 3 arterial oxygen tension PaO₂ over inspiratory oxygen fraction FIO₂ of less than 300 mm Hg. In the blood detection cohort Figure 1 A , patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them 100% were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them 23.5% were severe cases.",
"In the blood detection cohort Figure 1 A , patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them 100% were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them 23.5% were severe cases. The ratio of severe symptoms between these two groups was significantly different p value = 0.0001 . In the anal swab cohort Figure 1 B , 11 of 28 cases were detected to be anal swab positive, 8 of them 72.7% were with severe symptoms, which was significantly higher than that 4 23.5% of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 Figure 2 A was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing.",
"Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 Figure 2 A was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 Ct = 17 + 25 , 7, 8 Ct = 25 + 26 , and 11 Ct = 15 + 25 . In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal Ct = Neg+39 , and then the signal was gone again on day 8. On day 9, a low level of viral RNA Ct = 36 + 41 was detected again in the blood.",
"In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal Ct = Neg+39 , and then the signal was gone again on day 8. On day 9, a low level of viral RNA Ct = 36 + 41 was detected again in the blood. On day 12, the blood lost signal again. A high concentration of virus RNA Ct = 23 + 27 was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 Figure 2 B , who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes.",
"Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 Figure 2 B , who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 Ct = 34 + 36 and 8 Ct = 38 + 38 . His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus Ct = 25 + 36 were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal Ct = 23 + 24 , blood Ct = 34 + 39 and anal Ct = 24 + 29 samples on day 10.",
"Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus Ct = 25 + 36 were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal Ct = 23 + 24 , blood Ct = 34 + 39 and anal Ct = 24 + 29 samples on day 10. He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 Figure 3 A was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal Ct = 30 + 30 and blood samples Ct = 37 + 39 on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22.",
"Patient 3 Figure 3 A was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal Ct = 30 + 30 and blood samples Ct = 37 + 39 on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 Figure 3 B was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive Ct = 32 + 37 . On day 9, he was transferred out. Patient 5 Figure 3 C was admitted on illness day 4 and his blood sample was virus-positive Ct = 38 + Neg on day 6.",
"On day 9, he was transferred out. Patient 5 Figure 3 C was admitted on illness day 4 and his blood sample was virus-positive Ct = 38 + Neg on day 6. Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 Figure 3 D with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory.",
"As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in or later progressed to severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group p = 0.0001 . Patient 2 Figure 2 B , 5 Figure 3 C and 6 Figure 3 D all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 Figure 2 A and 3 Figure 3 A who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 Figure 3 B who had serum sample collected one day post the diagnosis of severe illness.",
"Patient 2 Figure 2 B , 5 Figure 3 C and 6 Figure 3 D all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 Figure 2 A and 3 Figure 3 A who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 Figure 3 B who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity p = 0.0102 . The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region.",
"The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA Ct = 23 + 27, on day 13 was detected in anal swab but not in pharyngeal the same day and blood 1 d ahead . For patient 2, higher concentrations of viral RNAs were detected in anal swab Ct = 24 + 39 and pharyngeal swab Ct = 23 + 24 than in the blood Ct = 34 + 39 on the same day.",
"For patient 1, a high concentration of viral RNA Ct = 23 + 27, on day 13 was detected in anal swab but not in pharyngeal the same day and blood 1 d ahead . For patient 2, higher concentrations of viral RNAs were detected in anal swab Ct = 24 + 39 and pharyngeal swab Ct = 23 + 24 than in the blood Ct = 34 + 39 on the same day. Angiotensin-converting enzyme 2 ACE2 still is one of the receptors for 2019-nCoV attachment and entry . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body.",
"Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool.",
"Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction."
] | 2,519 | 1,169 |
What test could give an indication for special care for 2019-nCOV patients? | presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care | [
"The novel coronavirus 2019-nCoV infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood 6 of 57 patients and the anal swabs 11 of 28 patients . Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity p-value = 0.0001 . Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab Ct value = 24 + 39 was higher than in the blood Ct value = 34 + 39 from patient 2, suggesting that the virus might replicate in the digestive tract.",
"Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab Ct value = 24 + 39 was higher than in the blood Ct value = 34 + 39 from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus 2019-nCoV , originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination , transmission and evolution , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence.",
"Basic scientific research has achieved significantly in the investigation of viral origination , transmission and evolution , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital.",
"Several studies have roughly described the overall clinical features of 2019-nCoV patients . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes.",
"In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit Da'an Gene Corporation, Cat: DA0630 on an automatic workstation Smart 32 Da'an Gene Corporation following the guidelines. Real-time reverse transcriptional polymerase chain reaction RT-PCR reagent Da'an Gene cooperation, Cat DA0930 was employed for viral detection per the protocol.",
"Viral RNA was extracted with Nucleic Acid Isolation Kit Da'an Gene Corporation, Cat: DA0630 on an automatic workstation Smart 32 Da'an Gene Corporation following the guidelines. Real-time reverse transcriptional polymerase chain reaction RT-PCR reagent Da'an Gene cooperation, Cat DA0930 was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab FAM reporter and N VIC reporter genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set s gave a reliable signal s . All patients had pneumonia-based diseases but with diversified clinical manifestation.",
"Samples were considered to be viral positive when either or both set s gave a reliable signal s . All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1 distress of respiratory with respiratory rate > = 30/min; 2 Oxygen saturation < = 93% in the rest state, and 3 arterial oxygen tension PaO₂ over inspiratory oxygen fraction FIO₂ of less than 300 mm Hg. In the blood detection cohort Figure 1 A , patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them 100% were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them 23.5% were severe cases.",
"In the blood detection cohort Figure 1 A , patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them 100% were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them 23.5% were severe cases. The ratio of severe symptoms between these two groups was significantly different p value = 0.0001 . In the anal swab cohort Figure 1 B , 11 of 28 cases were detected to be anal swab positive, 8 of them 72.7% were with severe symptoms, which was significantly higher than that 4 23.5% of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 Figure 2 A was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing.",
"Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 Figure 2 A was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 Ct = 17 + 25 , 7, 8 Ct = 25 + 26 , and 11 Ct = 15 + 25 . In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal Ct = Neg+39 , and then the signal was gone again on day 8. On day 9, a low level of viral RNA Ct = 36 + 41 was detected again in the blood.",
"In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal Ct = Neg+39 , and then the signal was gone again on day 8. On day 9, a low level of viral RNA Ct = 36 + 41 was detected again in the blood. On day 12, the blood lost signal again. A high concentration of virus RNA Ct = 23 + 27 was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 Figure 2 B , who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes.",
"Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 Figure 2 B , who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 Ct = 34 + 36 and 8 Ct = 38 + 38 . His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus Ct = 25 + 36 were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal Ct = 23 + 24 , blood Ct = 34 + 39 and anal Ct = 24 + 29 samples on day 10.",
"Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus Ct = 25 + 36 were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal Ct = 23 + 24 , blood Ct = 34 + 39 and anal Ct = 24 + 29 samples on day 10. He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 Figure 3 A was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal Ct = 30 + 30 and blood samples Ct = 37 + 39 on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22.",
"Patient 3 Figure 3 A was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal Ct = 30 + 30 and blood samples Ct = 37 + 39 on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 Figure 3 B was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive Ct = 32 + 37 . On day 9, he was transferred out. Patient 5 Figure 3 C was admitted on illness day 4 and his blood sample was virus-positive Ct = 38 + Neg on day 6.",
"On day 9, he was transferred out. Patient 5 Figure 3 C was admitted on illness day 4 and his blood sample was virus-positive Ct = 38 + Neg on day 6. Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 Figure 3 D with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory.",
"As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in or later progressed to severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group p = 0.0001 . Patient 2 Figure 2 B , 5 Figure 3 C and 6 Figure 3 D all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 Figure 2 A and 3 Figure 3 A who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 Figure 3 B who had serum sample collected one day post the diagnosis of severe illness.",
"Patient 2 Figure 2 B , 5 Figure 3 C and 6 Figure 3 D all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 Figure 2 A and 3 Figure 3 A who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 Figure 3 B who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity p = 0.0102 . The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region.",
"The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA Ct = 23 + 27, on day 13 was detected in anal swab but not in pharyngeal the same day and blood 1 d ahead . For patient 2, higher concentrations of viral RNAs were detected in anal swab Ct = 24 + 39 and pharyngeal swab Ct = 23 + 24 than in the blood Ct = 34 + 39 on the same day.",
"For patient 1, a high concentration of viral RNA Ct = 23 + 27, on day 13 was detected in anal swab but not in pharyngeal the same day and blood 1 d ahead . For patient 2, higher concentrations of viral RNAs were detected in anal swab Ct = 24 + 39 and pharyngeal swab Ct = 23 + 24 than in the blood Ct = 34 + 39 on the same day. Angiotensin-converting enzyme 2 ACE2 still is one of the receptors for 2019-nCoV attachment and entry . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body.",
"Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool.",
"Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction."
] | 2,519 | 1,171 |
What is the relationship between the presence of virus in anal swabs and disease severity in 2019-nCOV? | In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases | [
"The novel coronavirus 2019-nCoV infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood 6 of 57 patients and the anal swabs 11 of 28 patients . Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity p-value = 0.0001 . Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab Ct value = 24 + 39 was higher than in the blood Ct value = 34 + 39 from patient 2, suggesting that the virus might replicate in the digestive tract.",
"Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab Ct value = 24 + 39 was higher than in the blood Ct value = 34 + 39 from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus 2019-nCoV , originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination , transmission and evolution , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence.",
"Basic scientific research has achieved significantly in the investigation of viral origination , transmission and evolution , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital.",
"Several studies have roughly described the overall clinical features of 2019-nCoV patients . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes.",
"In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit Da'an Gene Corporation, Cat: DA0630 on an automatic workstation Smart 32 Da'an Gene Corporation following the guidelines. Real-time reverse transcriptional polymerase chain reaction RT-PCR reagent Da'an Gene cooperation, Cat DA0930 was employed for viral detection per the protocol.",
"Viral RNA was extracted with Nucleic Acid Isolation Kit Da'an Gene Corporation, Cat: DA0630 on an automatic workstation Smart 32 Da'an Gene Corporation following the guidelines. Real-time reverse transcriptional polymerase chain reaction RT-PCR reagent Da'an Gene cooperation, Cat DA0930 was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab FAM reporter and N VIC reporter genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set s gave a reliable signal s . All patients had pneumonia-based diseases but with diversified clinical manifestation.",
"Samples were considered to be viral positive when either or both set s gave a reliable signal s . All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1 distress of respiratory with respiratory rate > = 30/min; 2 Oxygen saturation < = 93% in the rest state, and 3 arterial oxygen tension PaO₂ over inspiratory oxygen fraction FIO₂ of less than 300 mm Hg. In the blood detection cohort Figure 1 A , patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them 100% were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them 23.5% were severe cases.",
"In the blood detection cohort Figure 1 A , patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them 100% were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them 23.5% were severe cases. The ratio of severe symptoms between these two groups was significantly different p value = 0.0001 . In the anal swab cohort Figure 1 B , 11 of 28 cases were detected to be anal swab positive, 8 of them 72.7% were with severe symptoms, which was significantly higher than that 4 23.5% of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 Figure 2 A was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing.",
"Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 Figure 2 A was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 Ct = 17 + 25 , 7, 8 Ct = 25 + 26 , and 11 Ct = 15 + 25 . In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal Ct = Neg+39 , and then the signal was gone again on day 8. On day 9, a low level of viral RNA Ct = 36 + 41 was detected again in the blood.",
"In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal Ct = Neg+39 , and then the signal was gone again on day 8. On day 9, a low level of viral RNA Ct = 36 + 41 was detected again in the blood. On day 12, the blood lost signal again. A high concentration of virus RNA Ct = 23 + 27 was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 Figure 2 B , who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes.",
"Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 Figure 2 B , who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 Ct = 34 + 36 and 8 Ct = 38 + 38 . His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus Ct = 25 + 36 were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal Ct = 23 + 24 , blood Ct = 34 + 39 and anal Ct = 24 + 29 samples on day 10.",
"Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus Ct = 25 + 36 were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal Ct = 23 + 24 , blood Ct = 34 + 39 and anal Ct = 24 + 29 samples on day 10. He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 Figure 3 A was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal Ct = 30 + 30 and blood samples Ct = 37 + 39 on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22.",
"Patient 3 Figure 3 A was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal Ct = 30 + 30 and blood samples Ct = 37 + 39 on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 Figure 3 B was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive Ct = 32 + 37 . On day 9, he was transferred out. Patient 5 Figure 3 C was admitted on illness day 4 and his blood sample was virus-positive Ct = 38 + Neg on day 6.",
"On day 9, he was transferred out. Patient 5 Figure 3 C was admitted on illness day 4 and his blood sample was virus-positive Ct = 38 + Neg on day 6. Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 Figure 3 D with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory.",
"As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in or later progressed to severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group p = 0.0001 . Patient 2 Figure 2 B , 5 Figure 3 C and 6 Figure 3 D all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 Figure 2 A and 3 Figure 3 A who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 Figure 3 B who had serum sample collected one day post the diagnosis of severe illness.",
"Patient 2 Figure 2 B , 5 Figure 3 C and 6 Figure 3 D all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 Figure 2 A and 3 Figure 3 A who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 Figure 3 B who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity p = 0.0102 . The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region.",
"The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA Ct = 23 + 27, on day 13 was detected in anal swab but not in pharyngeal the same day and blood 1 d ahead . For patient 2, higher concentrations of viral RNAs were detected in anal swab Ct = 24 + 39 and pharyngeal swab Ct = 23 + 24 than in the blood Ct = 34 + 39 on the same day.",
"For patient 1, a high concentration of viral RNA Ct = 23 + 27, on day 13 was detected in anal swab but not in pharyngeal the same day and blood 1 d ahead . For patient 2, higher concentrations of viral RNAs were detected in anal swab Ct = 24 + 39 and pharyngeal swab Ct = 23 + 24 than in the blood Ct = 34 + 39 on the same day. Angiotensin-converting enzyme 2 ACE2 still is one of the receptors for 2019-nCoV attachment and entry . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body.",
"Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool.",
"Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction."
] | 2,519 | 1,170 |
What could be the implication of 2019-nCOV virus in anal swabs? | digestive tract might be one extrapulmonary site for virus replication | [
"The novel coronavirus 2019-nCoV infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood 6 of 57 patients and the anal swabs 11 of 28 patients . Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity p-value = 0.0001 . Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab Ct value = 24 + 39 was higher than in the blood Ct value = 34 + 39 from patient 2, suggesting that the virus might replicate in the digestive tract.",
"Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab Ct value = 24 + 39 was higher than in the blood Ct value = 34 + 39 from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus 2019-nCoV , originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination , transmission and evolution , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence.",
"Basic scientific research has achieved significantly in the investigation of viral origination , transmission and evolution , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital.",
"Several studies have roughly described the overall clinical features of 2019-nCoV patients . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes.",
"In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit Da'an Gene Corporation, Cat: DA0630 on an automatic workstation Smart 32 Da'an Gene Corporation following the guidelines. Real-time reverse transcriptional polymerase chain reaction RT-PCR reagent Da'an Gene cooperation, Cat DA0930 was employed for viral detection per the protocol.",
"Viral RNA was extracted with Nucleic Acid Isolation Kit Da'an Gene Corporation, Cat: DA0630 on an automatic workstation Smart 32 Da'an Gene Corporation following the guidelines. Real-time reverse transcriptional polymerase chain reaction RT-PCR reagent Da'an Gene cooperation, Cat DA0930 was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab FAM reporter and N VIC reporter genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set s gave a reliable signal s . All patients had pneumonia-based diseases but with diversified clinical manifestation.",
"Samples were considered to be viral positive when either or both set s gave a reliable signal s . All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1 distress of respiratory with respiratory rate > = 30/min; 2 Oxygen saturation < = 93% in the rest state, and 3 arterial oxygen tension PaO₂ over inspiratory oxygen fraction FIO₂ of less than 300 mm Hg. In the blood detection cohort Figure 1 A , patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them 100% were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them 23.5% were severe cases.",
"In the blood detection cohort Figure 1 A , patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them 100% were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them 23.5% were severe cases. The ratio of severe symptoms between these two groups was significantly different p value = 0.0001 . In the anal swab cohort Figure 1 B , 11 of 28 cases were detected to be anal swab positive, 8 of them 72.7% were with severe symptoms, which was significantly higher than that 4 23.5% of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 Figure 2 A was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing.",
"Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 Figure 2 A was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 Ct = 17 + 25 , 7, 8 Ct = 25 + 26 , and 11 Ct = 15 + 25 . In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal Ct = Neg+39 , and then the signal was gone again on day 8. On day 9, a low level of viral RNA Ct = 36 + 41 was detected again in the blood.",
"In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal Ct = Neg+39 , and then the signal was gone again on day 8. On day 9, a low level of viral RNA Ct = 36 + 41 was detected again in the blood. On day 12, the blood lost signal again. A high concentration of virus RNA Ct = 23 + 27 was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 Figure 2 B , who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes.",
"Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 Figure 2 B , who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 Ct = 34 + 36 and 8 Ct = 38 + 38 . His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus Ct = 25 + 36 were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal Ct = 23 + 24 , blood Ct = 34 + 39 and anal Ct = 24 + 29 samples on day 10.",
"Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus Ct = 25 + 36 were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal Ct = 23 + 24 , blood Ct = 34 + 39 and anal Ct = 24 + 29 samples on day 10. He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 Figure 3 A was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal Ct = 30 + 30 and blood samples Ct = 37 + 39 on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22.",
"Patient 3 Figure 3 A was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal Ct = 30 + 30 and blood samples Ct = 37 + 39 on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 Figure 3 B was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive Ct = 32 + 37 . On day 9, he was transferred out. Patient 5 Figure 3 C was admitted on illness day 4 and his blood sample was virus-positive Ct = 38 + Neg on day 6.",
"On day 9, he was transferred out. Patient 5 Figure 3 C was admitted on illness day 4 and his blood sample was virus-positive Ct = 38 + Neg on day 6. Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 Figure 3 D with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory.",
"As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in or later progressed to severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group p = 0.0001 . Patient 2 Figure 2 B , 5 Figure 3 C and 6 Figure 3 D all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 Figure 2 A and 3 Figure 3 A who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 Figure 3 B who had serum sample collected one day post the diagnosis of severe illness.",
"Patient 2 Figure 2 B , 5 Figure 3 C and 6 Figure 3 D all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 Figure 2 A and 3 Figure 3 A who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 Figure 3 B who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity p = 0.0102 . The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region.",
"The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA Ct = 23 + 27, on day 13 was detected in anal swab but not in pharyngeal the same day and blood 1 d ahead . For patient 2, higher concentrations of viral RNAs were detected in anal swab Ct = 24 + 39 and pharyngeal swab Ct = 23 + 24 than in the blood Ct = 34 + 39 on the same day.",
"For patient 1, a high concentration of viral RNA Ct = 23 + 27, on day 13 was detected in anal swab but not in pharyngeal the same day and blood 1 d ahead . For patient 2, higher concentrations of viral RNAs were detected in anal swab Ct = 24 + 39 and pharyngeal swab Ct = 23 + 24 than in the blood Ct = 34 + 39 on the same day. Angiotensin-converting enzyme 2 ACE2 still is one of the receptors for 2019-nCoV attachment and entry . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body.",
"Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool.",
"Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction."
] | 2,519 | 1,172 |
What could account for the high transmission rate of the 2019-nCOV virus? | Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. | [
"The novel coronavirus 2019-nCoV infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood 6 of 57 patients and the anal swabs 11 of 28 patients . Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity p-value = 0.0001 . Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab Ct value = 24 + 39 was higher than in the blood Ct value = 34 + 39 from patient 2, suggesting that the virus might replicate in the digestive tract.",
"Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab Ct value = 24 + 39 was higher than in the blood Ct value = 34 + 39 from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus 2019-nCoV , originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination , transmission and evolution , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence.",
"Basic scientific research has achieved significantly in the investigation of viral origination , transmission and evolution , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital.",
"Several studies have roughly described the overall clinical features of 2019-nCoV patients . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes.",
"In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit Da'an Gene Corporation, Cat: DA0630 on an automatic workstation Smart 32 Da'an Gene Corporation following the guidelines. Real-time reverse transcriptional polymerase chain reaction RT-PCR reagent Da'an Gene cooperation, Cat DA0930 was employed for viral detection per the protocol.",
"Viral RNA was extracted with Nucleic Acid Isolation Kit Da'an Gene Corporation, Cat: DA0630 on an automatic workstation Smart 32 Da'an Gene Corporation following the guidelines. Real-time reverse transcriptional polymerase chain reaction RT-PCR reagent Da'an Gene cooperation, Cat DA0930 was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab FAM reporter and N VIC reporter genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set s gave a reliable signal s . All patients had pneumonia-based diseases but with diversified clinical manifestation.",
"Samples were considered to be viral positive when either or both set s gave a reliable signal s . All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1 distress of respiratory with respiratory rate > = 30/min; 2 Oxygen saturation < = 93% in the rest state, and 3 arterial oxygen tension PaO₂ over inspiratory oxygen fraction FIO₂ of less than 300 mm Hg. In the blood detection cohort Figure 1 A , patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them 100% were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them 23.5% were severe cases.",
"In the blood detection cohort Figure 1 A , patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them 100% were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them 23.5% were severe cases. The ratio of severe symptoms between these two groups was significantly different p value = 0.0001 . In the anal swab cohort Figure 1 B , 11 of 28 cases were detected to be anal swab positive, 8 of them 72.7% were with severe symptoms, which was significantly higher than that 4 23.5% of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 Figure 2 A was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing.",
"Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 Figure 2 A was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 Ct = 17 + 25 , 7, 8 Ct = 25 + 26 , and 11 Ct = 15 + 25 . In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal Ct = Neg+39 , and then the signal was gone again on day 8. On day 9, a low level of viral RNA Ct = 36 + 41 was detected again in the blood.",
"In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal Ct = Neg+39 , and then the signal was gone again on day 8. On day 9, a low level of viral RNA Ct = 36 + 41 was detected again in the blood. On day 12, the blood lost signal again. A high concentration of virus RNA Ct = 23 + 27 was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 Figure 2 B , who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes.",
"Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 Figure 2 B , who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 Ct = 34 + 36 and 8 Ct = 38 + 38 . His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus Ct = 25 + 36 were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal Ct = 23 + 24 , blood Ct = 34 + 39 and anal Ct = 24 + 29 samples on day 10.",
"Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus Ct = 25 + 36 were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal Ct = 23 + 24 , blood Ct = 34 + 39 and anal Ct = 24 + 29 samples on day 10. He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 Figure 3 A was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal Ct = 30 + 30 and blood samples Ct = 37 + 39 on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22.",
"Patient 3 Figure 3 A was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal Ct = 30 + 30 and blood samples Ct = 37 + 39 on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 Figure 3 B was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive Ct = 32 + 37 . On day 9, he was transferred out. Patient 5 Figure 3 C was admitted on illness day 4 and his blood sample was virus-positive Ct = 38 + Neg on day 6.",
"On day 9, he was transferred out. Patient 5 Figure 3 C was admitted on illness day 4 and his blood sample was virus-positive Ct = 38 + Neg on day 6. Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 Figure 3 D with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory.",
"As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in or later progressed to severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group p = 0.0001 . Patient 2 Figure 2 B , 5 Figure 3 C and 6 Figure 3 D all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 Figure 2 A and 3 Figure 3 A who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 Figure 3 B who had serum sample collected one day post the diagnosis of severe illness.",
"Patient 2 Figure 2 B , 5 Figure 3 C and 6 Figure 3 D all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 Figure 2 A and 3 Figure 3 A who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 Figure 3 B who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity p = 0.0102 . The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region.",
"The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA Ct = 23 + 27, on day 13 was detected in anal swab but not in pharyngeal the same day and blood 1 d ahead . For patient 2, higher concentrations of viral RNAs were detected in anal swab Ct = 24 + 39 and pharyngeal swab Ct = 23 + 24 than in the blood Ct = 34 + 39 on the same day.",
"For patient 1, a high concentration of viral RNA Ct = 23 + 27, on day 13 was detected in anal swab but not in pharyngeal the same day and blood 1 d ahead . For patient 2, higher concentrations of viral RNAs were detected in anal swab Ct = 24 + 39 and pharyngeal swab Ct = 23 + 24 than in the blood Ct = 34 + 39 on the same day. Angiotensin-converting enzyme 2 ACE2 still is one of the receptors for 2019-nCoV attachment and entry . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body.",
"Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool.",
"Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction."
] | 2,519 | 1,173 |
What could account for the dissemination of the 2019-nCOV virus across the whole body? | We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa | [
"The novel coronavirus 2019-nCoV infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood 6 of 57 patients and the anal swabs 11 of 28 patients . Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity p-value = 0.0001 . Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab Ct value = 24 + 39 was higher than in the blood Ct value = 34 + 39 from patient 2, suggesting that the virus might replicate in the digestive tract.",
"Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab Ct value = 24 + 39 was higher than in the blood Ct value = 34 + 39 from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus 2019-nCoV , originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination , transmission and evolution , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence.",
"Basic scientific research has achieved significantly in the investigation of viral origination , transmission and evolution , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital.",
"Several studies have roughly described the overall clinical features of 2019-nCoV patients . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes.",
"In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit Da'an Gene Corporation, Cat: DA0630 on an automatic workstation Smart 32 Da'an Gene Corporation following the guidelines. Real-time reverse transcriptional polymerase chain reaction RT-PCR reagent Da'an Gene cooperation, Cat DA0930 was employed for viral detection per the protocol.",
"Viral RNA was extracted with Nucleic Acid Isolation Kit Da'an Gene Corporation, Cat: DA0630 on an automatic workstation Smart 32 Da'an Gene Corporation following the guidelines. Real-time reverse transcriptional polymerase chain reaction RT-PCR reagent Da'an Gene cooperation, Cat DA0930 was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab FAM reporter and N VIC reporter genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set s gave a reliable signal s . All patients had pneumonia-based diseases but with diversified clinical manifestation.",
"Samples were considered to be viral positive when either or both set s gave a reliable signal s . All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1 distress of respiratory with respiratory rate > = 30/min; 2 Oxygen saturation < = 93% in the rest state, and 3 arterial oxygen tension PaO₂ over inspiratory oxygen fraction FIO₂ of less than 300 mm Hg. In the blood detection cohort Figure 1 A , patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them 100% were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them 23.5% were severe cases.",
"In the blood detection cohort Figure 1 A , patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them 100% were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them 23.5% were severe cases. The ratio of severe symptoms between these two groups was significantly different p value = 0.0001 . In the anal swab cohort Figure 1 B , 11 of 28 cases were detected to be anal swab positive, 8 of them 72.7% were with severe symptoms, which was significantly higher than that 4 23.5% of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 Figure 2 A was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing.",
"Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 Figure 2 A was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 Ct = 17 + 25 , 7, 8 Ct = 25 + 26 , and 11 Ct = 15 + 25 . In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal Ct = Neg+39 , and then the signal was gone again on day 8. On day 9, a low level of viral RNA Ct = 36 + 41 was detected again in the blood.",
"In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal Ct = Neg+39 , and then the signal was gone again on day 8. On day 9, a low level of viral RNA Ct = 36 + 41 was detected again in the blood. On day 12, the blood lost signal again. A high concentration of virus RNA Ct = 23 + 27 was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 Figure 2 B , who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes.",
"Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 Figure 2 B , who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 Ct = 34 + 36 and 8 Ct = 38 + 38 . His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus Ct = 25 + 36 were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal Ct = 23 + 24 , blood Ct = 34 + 39 and anal Ct = 24 + 29 samples on day 10.",
"Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus Ct = 25 + 36 were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal Ct = 23 + 24 , blood Ct = 34 + 39 and anal Ct = 24 + 29 samples on day 10. He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 Figure 3 A was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal Ct = 30 + 30 and blood samples Ct = 37 + 39 on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22.",
"Patient 3 Figure 3 A was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal Ct = 30 + 30 and blood samples Ct = 37 + 39 on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 Figure 3 B was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive Ct = 32 + 37 . On day 9, he was transferred out. Patient 5 Figure 3 C was admitted on illness day 4 and his blood sample was virus-positive Ct = 38 + Neg on day 6.",
"On day 9, he was transferred out. Patient 5 Figure 3 C was admitted on illness day 4 and his blood sample was virus-positive Ct = 38 + Neg on day 6. Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 Figure 3 D with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory.",
"As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in or later progressed to severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group p = 0.0001 . Patient 2 Figure 2 B , 5 Figure 3 C and 6 Figure 3 D all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 Figure 2 A and 3 Figure 3 A who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 Figure 3 B who had serum sample collected one day post the diagnosis of severe illness.",
"Patient 2 Figure 2 B , 5 Figure 3 C and 6 Figure 3 D all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 Figure 2 A and 3 Figure 3 A who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 Figure 3 B who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity p = 0.0102 . The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region.",
"The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA Ct = 23 + 27, on day 13 was detected in anal swab but not in pharyngeal the same day and blood 1 d ahead . For patient 2, higher concentrations of viral RNAs were detected in anal swab Ct = 24 + 39 and pharyngeal swab Ct = 23 + 24 than in the blood Ct = 34 + 39 on the same day.",
"For patient 1, a high concentration of viral RNA Ct = 23 + 27, on day 13 was detected in anal swab but not in pharyngeal the same day and blood 1 d ahead . For patient 2, higher concentrations of viral RNAs were detected in anal swab Ct = 24 + 39 and pharyngeal swab Ct = 23 + 24 than in the blood Ct = 34 + 39 on the same day. Angiotensin-converting enzyme 2 ACE2 still is one of the receptors for 2019-nCoV attachment and entry . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body.",
"Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool.",
"Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction."
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When did the World Health Organization declare the Ebola epidemic in West Africa as a Public Health Emergency of International Concern? | 8 August 2014 | [
"Background: The Ebola epidemic in West Africa caused global fear and stirred up worldwide preparedness activities in countries sharing borders with those affected, and in geographically far-away countries such as Iceland. Objective: To describe and analyse Ebola preparedness activities within the Icelandic healthcare system, and to explore the perspectives and experiences of managers and frontline health workers. Methods: A qualitative case study, based on semi-structured interviews with 21 staff members in the national Ebola Treatment Team, Emergency Room at Landspitali University Hospital, and managers of the response team. Results: Contextual factors such as culture and demography influenced preparedness, and contributed to the positive state of mind of participants, and ingenuity in using available resources for preparedness. While participants believed they were ready to take on the task of Ebola, they also had doubts about the chances of Ebola ever reaching Iceland. Yet, factors such as fear of Ebola and the perceived stigma associated with caring for a potentially infected Ebola patient, influenced the preparation process and resulted in plans for specific precautions by staff to secure the safety of their families.",
"While participants believed they were ready to take on the task of Ebola, they also had doubts about the chances of Ebola ever reaching Iceland. Yet, factors such as fear of Ebola and the perceived stigma associated with caring for a potentially infected Ebola patient, influenced the preparation process and resulted in plans for specific precautions by staff to secure the safety of their families. There were also concerns about the teamwork and lack of commitment by some during training. Being a ‘tiny’ nation was seen as both an asset and a weakness in the preparation process. Honest information sharing and scenario-based training contributed to increased confidence amongst participants in the response plans. Conclusions: Communication and training were important for preparedness of health staff in Iceland, in order to receive, admit, and treat a patient suspected of having Ebola, while doubts prevailed on staff capacity to properly do so.",
"Honest information sharing and scenario-based training contributed to increased confidence amongst participants in the response plans. Conclusions: Communication and training were important for preparedness of health staff in Iceland, in order to receive, admit, and treat a patient suspected of having Ebola, while doubts prevailed on staff capacity to properly do so. For optimal preparedness, likely scenarios for future global security health threats need to be repeatedly enacted, and areas plagued by poverty and fragile healthcare systems require global support. Text: Global health; prevention and control; public policy; qualitative evaluation; emergency responders; communicable diseases; emerging; fear Background On 8 August 2014, the World Health Organization declared the Ebola epidemic in West Africa as a Public Health Emergency of International Concern PHEIC under the International Health Regulations IHR . All three of the worst affected countries were to address the emerging epidemic challenge without staff, stuff, space and systems . With the epidemic seemingly out of control, and a proportionately high number of doctors, nurses, and midwives succumbing to Ebola , there was a growing fear of transmission beyond the region.",
"All three of the worst affected countries were to address the emerging epidemic challenge without staff, stuff, space and systems . With the epidemic seemingly out of control, and a proportionately high number of doctors, nurses, and midwives succumbing to Ebola , there was a growing fear of transmission beyond the region. In breach of WHO recommendations and guidelines , flights were cancelled and cross-border movement curtailed . The epidemic caused public concern outside West Africa , as fear and racism found fertile ground , and in an effort to stop the international spread of the disease, all states were advised to be prepared to detect, investigate, and manage Ebola cases . Preparedness as part of disaster risk reduction is defined as 'the knowledge and capacities developed by governments, response and recovery organizations, communities and individuals to effectively anticipate, respond to, and recover from the impacts of likely, imminent or current disasters' . Yet, preparedness is also enveloped in and influenced by the socio-cultural dimension at the individual, organizational, and national levels, and measures to manage outbreaks are not always accepted or accommodated by the communities to which they are applied .",
"Preparedness as part of disaster risk reduction is defined as 'the knowledge and capacities developed by governments, response and recovery organizations, communities and individuals to effectively anticipate, respond to, and recover from the impacts of likely, imminent or current disasters' . Yet, preparedness is also enveloped in and influenced by the socio-cultural dimension at the individual, organizational, and national levels, and measures to manage outbreaks are not always accepted or accommodated by the communities to which they are applied . An analysis of eight European countries' preparedness plans since 2009 for countering a future influenza A H1N1 pandemic revealed that the way plans were framed varied considerably, and ' told us something about how the different countries want pandemics and preparedness to be understood by the public' . More research was encouraged into cultural and social structures in the respective countries. In Iceland, information about the Ebola epidemic in West Africa came from several sources. The Directorate of Health DH first reported on the epidemic on 8 April 2014 .",
"In Iceland, information about the Ebola epidemic in West Africa came from several sources. The Directorate of Health DH first reported on the epidemic on 8 April 2014 . In Icelandic media, the rapid progress of the Ebola epidemic in West Africa was increasingly highlighted, and exported Ebola cases to Spain, USA, and elsewhere, were widely covered. Fear of a global epidemic was rife, and in media and online discussions, doubts were raised about the Icelandic health system´s capacity to take care of a patient with Ebola , despite its ranking as one of the best in the world . On 11 August 2014, three days after WHO declared PHEIC because of Ebola, DH encouraged Icelandic citizens to avoid visits to the area, if possible, and reported that the national epidemic preparedness plan was being activated for Ebola . It was elaborated by a team that involved the Chief Epidemiologist at the DH, Landspitali University Hospital LSH , the Department of Civil Protection and Emergency Management DCPEM , and the seven Primary Healthcare Regional Organizations in the country at the time.",
"On 11 August 2014, three days after WHO declared PHEIC because of Ebola, DH encouraged Icelandic citizens to avoid visits to the area, if possible, and reported that the national epidemic preparedness plan was being activated for Ebola . It was elaborated by a team that involved the Chief Epidemiologist at the DH, Landspitali University Hospital LSH , the Department of Civil Protection and Emergency Management DCPEM , and the seven Primary Healthcare Regional Organizations in the country at the time. Key external partners were the European Centre for Disease Prevention and Control ECDC and WHO, in addition to Nordic collaborators in epidemic preparedness . At the same time, it was regarded as highly unlikely that Ebola Virus Disease EVD would spread in the country . Recognized scenarios included the possible appearance of an infected person in need of treatment, who could be either an Icelandic citizen who had visited or worked in one of the affected West African countries, or a person with signs of EVD on a trans-Atlantic flight in the navigation area controlled by Icelandic authorities . On 3 November 2014, the plan was put to the test when a foreign airline made a non-scheduled landing at Keflavík International Airport due to fear of EVD in one passenger from South Africa.",
"Recognized scenarios included the possible appearance of an infected person in need of treatment, who could be either an Icelandic citizen who had visited or worked in one of the affected West African countries, or a person with signs of EVD on a trans-Atlantic flight in the navigation area controlled by Icelandic authorities . On 3 November 2014, the plan was put to the test when a foreign airline made a non-scheduled landing at Keflavík International Airport due to fear of EVD in one passenger from South Africa. Parked in a closed-off area, a physician in full Personal Protective Equipment PPE entered the plane, but quickly ruled out Ebola . Irrespective of good or bad overall performance, health systems are tested in times of crisis, such as epidemics. Here, the aim is to describe and analyse the process of establishing preparedness plans for Ebola in Iceland, with a specific focus on the perspectives and experiences of managers and frontline health workers involved in the process. This study is part of a larger study on the impact that the global threat of the Ebola epidemic had in Iceland .",
"Here, the aim is to describe and analyse the process of establishing preparedness plans for Ebola in Iceland, with a specific focus on the perspectives and experiences of managers and frontline health workers involved in the process. This study is part of a larger study on the impact that the global threat of the Ebola epidemic had in Iceland . Qualitative case study methodology was applied, perceiving the preparedness planning and training process as the case with clear boundaries of the initiation, process, and wrap-up of preparedness planning and training. The study was conducted in April-May 2016, and the interviewed participants were administrators and frontline health professionals central to the case, so as to explore their perspectives and experiences concerning Ebola preparedness . Staff in managerial positions were contacted by one of the authors GG for permission to interview them based on their role in the preparedness plan. To identify potential interviewees in the Ebola Treatment Team ETT , the director of the team listed relevant email contacts.",
"Staff in managerial positions were contacted by one of the authors GG for permission to interview them based on their role in the preparedness plan. To identify potential interviewees in the Ebola Treatment Team ETT , the director of the team listed relevant email contacts. Those who responded positively were subsequently invited for an interview, conducted in Icelandic by one of the authors ÍEH , a physiotherapist. In case interviewees suggested other potential participants, they were invited through email to participate. A similar methodology was applied to identify participants from the Emergency Room ER . They were included in order to represent frontline health workers who worked in the only ER in Reykjavík, where persons exposed to EVD were most likely to first seek care in case of acute illness.",
"A similar methodology was applied to identify participants from the Emergency Room ER . They were included in order to represent frontline health workers who worked in the only ER in Reykjavík, where persons exposed to EVD were most likely to first seek care in case of acute illness. Three separate interview guides were developedone each for managers, ETT, and ER respectively see supplementary material . The interviews included open questions probing the role of their institution in preparedness, the experience of the training process, challenges encountered or expected, and any dilemmas that they may have experienced in relation to the preparedness plan. The recruitment of participants was concluded when saturation was reached. Each interview was recorded and took about 20 to 60 minutes; they were then transcribed and analysed using thematic analysis.",
"The recruitment of participants was concluded when saturation was reached. Each interview was recorded and took about 20 to 60 minutes; they were then transcribed and analysed using thematic analysis. The data material was read through repeatedly, sorted, and categorized, based on the participants' priorities in the representation of their views. From this exercise, three broad themes were inductively identified that corresponded to critical perspectives introduced by the participants. Permission to conduct the study was granted by Iceland's National Bioethics Committee VSN- and Landspitali University Hospital LSH 13-16, 4 February 2016 . Reporting on the results was guided by the COREC guidelines ; however, to ensure anonymity of the respondents within the small community of staff who took part in the preparedness activities, participant information is not associated to quotations.",
"Permission to conduct the study was granted by Iceland's National Bioethics Committee VSN- and Landspitali University Hospital LSH 13-16, 4 February 2016 . Reporting on the results was guided by the COREC guidelines ; however, to ensure anonymity of the respondents within the small community of staff who took part in the preparedness activities, participant information is not associated to quotations. The Icelandic Ebola Preparedness Plan included the establishment of an ETT within LSH , and the preparatory activities engaged more than two hundred staff across all of its departments. The ETT consisted of about 50 healthcare professionals who had volunteered to participate, including 11 doctors and 28 nurses, a few laboratory technicians, radiologists, and auxiliary nurses. They attended special training sessions focused on protocols for admission and treatment of a patient with EVD, the donning/doffing of PPE, and personal protective measures during patient care. A new provisory unit was designed to be set up on the ground floor to minimize the risk of infection spreading to other units within the hospital, with two rooms specifically identified for the care of a patient with EVD .",
"They attended special training sessions focused on protocols for admission and treatment of a patient with EVD, the donning/doffing of PPE, and personal protective measures during patient care. A new provisory unit was designed to be set up on the ground floor to minimize the risk of infection spreading to other units within the hospital, with two rooms specifically identified for the care of a patient with EVD . Managers' accounts of this period elaborated the complexity of preparedness planning in terms of the involved institutions, actors, procedures and requirement of the plan. One manager concluded: You get no discount. You can never go the shorter way. There was always something that surprised you.",
"One manager concluded: You get no discount. You can never go the shorter way. There was always something that surprised you. We thought this was a lot like a three headed monster, so when you chopped off one of its heads, three other emerged, every solution was followed by more problems. The health professionals who volunteered to join ETT did so for different reasons. Ebola preparedness was 'a job that had to be done', and 'someone had to do it'.",
"The health professionals who volunteered to join ETT did so for different reasons. Ebola preparedness was 'a job that had to be done', and 'someone had to do it'. Some referred to ethical or professional obligations: This is just a part of being a nurse, to encounter situations that can be dangerous to you or someone else, but you have made this decision and you deal with it. Some connected their decision to their 'action gene' or 'addiction to taking risks', while others said they had already raised their kids and had years of experience, including work with other epidemics, such as HIV. Yet, the practice of volunteering in the preparation was questioned. One participant said: We learned that we could not rely on volunteers … when you work in an infectious disease department you cannot choose what infections you want to work with.",
"Yet, the practice of volunteering in the preparation was questioned. One participant said: We learned that we could not rely on volunteers … when you work in an infectious disease department you cannot choose what infections you want to work with. ER staff indicated that for them working in the ER was enough of a risk to take, no reason to expose oneself even more by joining the ETT, and appreciated that others had volunteered. All participants noted that co-operation and communication had generally functioned well during the preparedness planning, with information flowing both ways. Short communication lines within the healthcare system were perceived as both a strength and a weakness; a strength, insofar as people knew each other, but a weakness because of the uneven burden of workload. Staff of the ETT and in the ER felt they had been well-informed, and that openness and honesty had characterized the planning and diminished their initial fear.",
"Short communication lines within the healthcare system were perceived as both a strength and a weakness; a strength, insofar as people knew each other, but a weakness because of the uneven burden of workload. Staff of the ETT and in the ER felt they had been well-informed, and that openness and honesty had characterized the planning and diminished their initial fear. Those in managerial positions had listened and taken their opinions into consideration. One said: They were honest, no one was hiding anything, everything was on the table, no one tried to make things more appealing and say that everything would be OK, they just told us about things as they were. Both management and participants from the ETT and ER expressed their ambiguity in terms of trust, doubt, and fear. Participants conveyed trust in the health system and their own role as health professionals, while at the same time admitting to facing formidable challenges during the elaboration of the preparedness plan.",
"Both management and participants from the ETT and ER expressed their ambiguity in terms of trust, doubt, and fear. Participants conveyed trust in the health system and their own role as health professionals, while at the same time admitting to facing formidable challenges during the elaboration of the preparedness plan. Facilities for isolation and treatment of patients with Ebola were less than perfect: We assessed how we could use the department … and change it in just a few hours into some kind of an isolation unit that we could possibly use. Some compared this short-term isolation facility to a 'camping site', as the facilities were too provisional and not comparable to those found elsewhere. There was also doubt about how many Ebola patients LSH would be able to care for: 'Maybe one or two patients, barely more'. Respondents believed that the training and education of the members of the ETT and ER had been satisfactory.",
"There was also doubt about how many Ebola patients LSH would be able to care for: 'Maybe one or two patients, barely more'. Respondents believed that the training and education of the members of the ETT and ER had been satisfactory. They felt that it had been proportionate to the risk, while some were concerned about the lack of staff. Nonetheless, there were contradictions on the division of labour among the professionals, exemplified by different ideas on how to proceed if a patient suspected of having an EVD came in an ambulance to the LSH for treatment. Almost all participants stated that they were ready to do their part in the Ebola response, or 'as ready as we could be'. There were diverse opinions on what it meant to be ready: to treat one confirmed case of Ebola, one suspected case, or more EVD patients?",
"Almost all participants stated that they were ready to do their part in the Ebola response, or 'as ready as we could be'. There were diverse opinions on what it meant to be ready: to treat one confirmed case of Ebola, one suspected case, or more EVD patients? When asked if Ebola was a real threat to the country, participants usually referred to how easy it was to travel the globe: 'Yeah, why not, the world is getting smaller'. Although Ebola was thought of as a real danger by many, some participants expressed difficulty in taking their training seriously, doubting that Ebola would ever reach Iceland. One respondent said: People were dedicated in the beginning, but when the news appeared that Ebola was receding, that diminished, and I never felt like this formally ended. Participants described their relief that nothing really happened, while emphasizing the need to experience a real situation to evaluate the preparedness efforts.",
"One respondent said: People were dedicated in the beginning, but when the news appeared that Ebola was receding, that diminished, and I never felt like this formally ended. Participants described their relief that nothing really happened, while emphasizing the need to experience a real situation to evaluate the preparedness efforts. One participant said that 'a little bit more seriousness would have been needed in the PPE practices'. It was taken as a manifestation of fear that some of the staff in the communicable disease department of the LSH refused to take part in the ETT. When describing their fears, ETT members frequently connected it to their working conditions. Many of them were afraid that they would not get the best PPE, others that they would not do the donning/doffing correctly and, lastly, they were worried about work performance while in the PPE.",
"When describing their fears, ETT members frequently connected it to their working conditions. Many of them were afraid that they would not get the best PPE, others that they would not do the donning/doffing correctly and, lastly, they were worried about work performance while in the PPE. One participant said: What bothered most of us was how uncomfortable the PPE was and I think that made people nervous: \"How will I manage working in this for hours?\" Another described the donning/doffing process like a 'complicated ballroom dance'. Moreover, participants were afraid of 'unknown territories', that is, they did not know the hospital ward, they were supposed to work in, and some team members had no recent experience of clinical work. One participant said: I didn't think these non-clinical people belonged in the team, because this is a very clinical environment in addition to having to be in this costume PPE with the risk of becoming infected by mistake.",
"Moreover, participants were afraid of 'unknown territories', that is, they did not know the hospital ward, they were supposed to work in, and some team members had no recent experience of clinical work. One participant said: I didn't think these non-clinical people belonged in the team, because this is a very clinical environment in addition to having to be in this costume PPE with the risk of becoming infected by mistake. Those with non-clinical background were, however, aware of their limitations: I realized that I would not be the one in the front, I would not be managing patients directly. The importance ascribed to teamwork was evident in relation to fear. Participants described fear of working with people they had not worked with before: The weakest link in the preparation was that even though I knew their faces, I had never worked with them. Another issue was no-show by some team members in training sessions or in lectures: This is team-work, one does this and the other one does this, we help each other.",
"Participants described fear of working with people they had not worked with before: The weakest link in the preparation was that even though I knew their faces, I had never worked with them. Another issue was no-show by some team members in training sessions or in lectures: This is team-work, one does this and the other one does this, we help each other. Then you don't want to be working with someone who didn't show up. There were a lot of doctors who just dropped in, dropped out, and then dropped in again. I asked myself: Are these individuals … ready to take this on? Participants in the ETT mentioned the precautions they took or intended to take to cope with their feelings of fear, should Ebola emerge in Iceland.",
"I asked myself: Are these individuals … ready to take this on? Participants in the ETT mentioned the precautions they took or intended to take to cope with their feelings of fear, should Ebola emerge in Iceland. A major precaution was planning to avoid contact with the family while working with Ebola patients. One participant said: 'You thought … about your children at school … parents in the neighbourhood …' if they knew s he was working with an Ebola patient. For them, it was important they would have access to special accommodation in case of clinical EVD work 'so I wouldn't be exposing anyone or creating hysteria'. ETT members mentioned the extra insurance offered as a prerequisite for taking part in the team.",
"For them, it was important they would have access to special accommodation in case of clinical EVD work 'so I wouldn't be exposing anyone or creating hysteria'. ETT members mentioned the extra insurance offered as a prerequisite for taking part in the team. 'The normal insurance for LHS staff would not cover everything if we were to become sick or even lose our lives.' Amongst ER staff, the matter of insurance did seem to be less of an issue compared to the ETT. One respondent said: 'You are used to being at risk by many disease threats'. Furthermore, the issue of higher salaries and risk commission came up in the interviews, but overall did not matter as much to the participants as the insurance, or assurance of accommodation in case of need.",
"One respondent said: 'You are used to being at risk by many disease threats'. Furthermore, the issue of higher salaries and risk commission came up in the interviews, but overall did not matter as much to the participants as the insurance, or assurance of accommodation in case of need. Characteristics associated with Iceland and the Icelandic people were referred to repeatedly by participants. The concept 'Tiny Iceland' was often mentioned and emerged with positive and negative connotations. 'Tiny Iceland' referred to the size of the country and population and its perceived capability to still 'get the job done'. even though compromises had to be made.",
"'Tiny Iceland' referred to the size of the country and population and its perceived capability to still 'get the job done'. even though compromises had to be made. Comparing how Iceland handled its responsibilities differently from other countries of a larger size was often brought up, both with pride in Iceland as a strong independent nation, and with insecurities about its capacity in comparison to other countries. It was pointed out that since the preparedness process was in the hands of a few people, everyone knew their role. As one administrator said: This little hospital system, as complicated as it might seem every day, gives you the chance to just pick up the phone and call the one in charge. Being a small population presents challenges regarding resources, infrastructure, and specialized medical training to comply with standards of international actors.",
"As one administrator said: This little hospital system, as complicated as it might seem every day, gives you the chance to just pick up the phone and call the one in charge. Being a small population presents challenges regarding resources, infrastructure, and specialized medical training to comply with standards of international actors. Notions of Icelanders as resilient in spite of shortcomings were common; referring to the experience of preparedness planning and training, one health staff said: It was very much the Icelandic way, we'll manage, we'll work it out, and there was so much ingenuity. This notion of a particular Icelandic approach to coping, in spite of shortcomings, was also detected more generally, as in the statement: Would it have worked? Yes, it would have worked. Would it have been optimal?",
"Yes, it would have worked. Would it have been optimal? We cannot say, it would have been optimal; we can say, it would have been sufficient. In contrast to this, there were concerns about whether Icelandic aid workers falling ill in Ebolaaffected countries should be transferred to Iceland or to hospitals in other Nordic countries with better isolation units. Some of the participants trusted that patients with EVD would not be transferred to Iceland. One participant stated: You heard that Norwegians were criticized for transferring their aid worker from Africa to Norway. We don't know what would have happened if they would have transferred an Icelander into the country.",
"One participant stated: You heard that Norwegians were criticized for transferring their aid worker from Africa to Norway. We don't know what would have happened if they would have transferred an Icelander into the country. We don't have good enough isolation unitsyou are not supposed to send patients to a hospital that is less than 100%. I thought there was assurance in that. During the devastating Ebola epidemic in West Africa that spread to neighbouring sub-Saharan countries, North America, and Europe , preparedness plans were widely elaborated and later evaluated. Evaluations have, for example, been conducted in 11 African countries close to the epidemic , in the EU region , and the US .",
"During the devastating Ebola epidemic in West Africa that spread to neighbouring sub-Saharan countries, North America, and Europe , preparedness plans were widely elaborated and later evaluated. Evaluations have, for example, been conducted in 11 African countries close to the epidemic , in the EU region , and the US . Here we present data from a qualitative case study on the process, and experiences with establishing a preparedness plan for Ebola in Iceland in 2014. Interviews with staff who were engaged, either as administrators or frontline healthcare workers, alert us to the manner in which geographic, demographic, cultural, and organizational characteristics shaped the response. The results show that the process of establishing and training for preparedness was permeated by ambiguities of pride and pragmatism, trust, doubts, and fear. 'Getting the job done' theme 1 refers to the multitude of tasks and considerations that surrounds and feeds into the preparedness plan itself and are necessary for successful planning and implementation.",
"The results show that the process of establishing and training for preparedness was permeated by ambiguities of pride and pragmatism, trust, doubts, and fear. 'Getting the job done' theme 1 refers to the multitude of tasks and considerations that surrounds and feeds into the preparedness plan itself and are necessary for successful planning and implementation. Using the metaphors of 'hard core' and 'soft periphery', Langley and Denis emphasize the importance of relatively 'peripheral' concerns and processes for planning and implementation of new interventions. The hard core represents the actual intervention or goal, e.g. implementation of a preparedness plan. The soft periphery refers to all the contextually important networking, negotiations, and agreements necessary to deliver the hard core.",
"implementation of a preparedness plan. The soft periphery refers to all the contextually important networking, negotiations, and agreements necessary to deliver the hard core. If the soft periphery is neglected, it will cause multiple challenges in the implementation process, and the benefit of the hard core, the intervention itself, may not transpire as anticipated. Due attention to the soft periphery may, however, considerably promote the delivery of an innovation, and secure support from important stakeholders. In our data, one manager speaks of the preparedness process as dealing with a three-headed monster where every solution was followed by new problems. The data indicate that the process of dealing with 'the three headed monster' was given due attention as a means to successfully develop Iceland's preparedness plan.",
"In our data, one manager speaks of the preparedness process as dealing with a three-headed monster where every solution was followed by new problems. The data indicate that the process of dealing with 'the three headed monster' was given due attention as a means to successfully develop Iceland's preparedness plan. Comprehensive consultations and the involvement of many associated institutions were mentioned. Still ambiguity remained with some staff in terms of division of responsibilities and taskse.g. when transporting a patient potentially infected with Ebola from the airport to the hospital, and other such activities. During epidemics, rumours, gossip, and unreliable information on the news and social media spread rapidly, resulting in so-called 'infodemics' .",
"when transporting a patient potentially infected with Ebola from the airport to the hospital, and other such activities. During epidemics, rumours, gossip, and unreliable information on the news and social media spread rapidly, resulting in so-called 'infodemics' . The West African Ebola epidemic was covered widely by media , and the fear of Ebola reached every corner of the world, exemplified by travel bans from affected countries, and trade barriers , in contrast to the ongoing epidemic in the Democratic Republic of Congo . In our second theme, trust, doubt, and fear of health workers were represented. Although all intentions were good, concerns remained about the suitability and safety of the isolation ward, the PPE, and other tools, as well as adequate engagement of colleagues who might potentially work alongside them, in case an Ebola patient came to Iceland. The foreignness of putting on, removing, and working from within a PPE and the trustworthiness of available PPE were mentioned.",
"Although all intentions were good, concerns remained about the suitability and safety of the isolation ward, the PPE, and other tools, as well as adequate engagement of colleagues who might potentially work alongside them, in case an Ebola patient came to Iceland. The foreignness of putting on, removing, and working from within a PPE and the trustworthiness of available PPE were mentioned. In preparedness efforts in other countries, scarcity of resources in relation to manpower demand and problems with training and protocols involving PPE were common challenges . Similar problems were encountered in Iceland. Provisory treatment facility had to be designed, called 'camping site' by some, in contrast to facilities found elsewhere . Further, the ETT was established based on voluntary recruitment rather than on the staff's assigned roles within the healthcare system, a procedure that was deemed less than optimal.",
"Provisory treatment facility had to be designed, called 'camping site' by some, in contrast to facilities found elsewhere . Further, the ETT was established based on voluntary recruitment rather than on the staff's assigned roles within the healthcare system, a procedure that was deemed less than optimal. The members of the ETT pointed out that they had never worked together as a team under circumstances that demanded strict adherence to infectious control procedures. This eroded trust, compounded by the laissez-faire attitude of some of its members during the preparation exercises, possibly due to other competing tasks in a busy hospital and insufficient resources that hampered full participation . Further, it was a constraint that simulation exercises were not an option, found to be an important element in preparation for epidemics . This might have resulted in less than optimal staff protection for those who would have been in direct contact with an infected patient, as reported during the SARS epidemic in Canada .",
"Further, it was a constraint that simulation exercises were not an option, found to be an important element in preparation for epidemics . This might have resulted in less than optimal staff protection for those who would have been in direct contact with an infected patient, as reported during the SARS epidemic in Canada . Anthropological work on emergency preparedness emphasizes the connectedness between health professionals, technological devices, and knowledge as a prerequisite for successful preparedness. Wolf and Hall present preparedness efforts as a form of governance that involves human bodies those of health professionals , clinical architectures e.g. isolation wards , and technical artefacts gloves, protective suits, disinfectants, etc. .",
"isolation wards , and technical artefacts gloves, protective suits, disinfectants, etc. . During preparedness training and implementation, 'nursing bodies are transformed into instruments of preparedness', and become part of infrastructural arrangements. Health professionals are, here, both vulnerable and powerful tools in the management of contamination. The authors argue that successful planning, training, and implementation of a preparedness plan require such intrinsic connectedness. In the case of Ebola preparedness in Iceland, health professionals draw our attention to dilemmas of connectedness, and their assessment of the fact that these shortcomings might hamper the mobilization of 'preparedness within the human body'that is, the embodied experience, routine, and tacit knowledge which Wolf and Hall state are key to successful implementation.",
"The authors argue that successful planning, training, and implementation of a preparedness plan require such intrinsic connectedness. In the case of Ebola preparedness in Iceland, health professionals draw our attention to dilemmas of connectedness, and their assessment of the fact that these shortcomings might hamper the mobilization of 'preparedness within the human body'that is, the embodied experience, routine, and tacit knowledge which Wolf and Hall state are key to successful implementation. Repeated enactment of receiving and treating a patient with Ebola within experienced and trustful teams would probably enhance such embodiment, provided that there is justified trust in the involved technology. In addition, repetition would also strengthen the 'soft periphery' of preparedness, and divisions of responsibilities would be clearer manifested. In the third theme, we observe how notions of the 'Icelandic way' help participants make sense of ambiguities about Ebola preparedness. Loftsdóttir explored how people negotiated the imagination of the local and the global during the 2008 economic crisis in Iceland .",
"In the third theme, we observe how notions of the 'Icelandic way' help participants make sense of ambiguities about Ebola preparedness. Loftsdóttir explored how people negotiated the imagination of the local and the global during the 2008 economic crisis in Iceland . Notions of the intrinsic character of Iceland, and of being Icelandic, serve to underscore certain points and explain positive and negative experiences with the preparedness plan. Iceland is far away from the continents, but still connected through global needs for policy, risk of contamination, and dependency in terms of collaboration, in emergencies emerging from elsewhere. In our study, participants highlighted the importance of believing in oneself and the 'Icelandic way of doing things,' summed up in the paraphrase 'þetta reddast' things always have a way of working out in the end . The preparedness plan had to be completed, and adapted to Iceland's particular global situation.",
"In our study, participants highlighted the importance of believing in oneself and the 'Icelandic way of doing things,' summed up in the paraphrase 'þetta reddast' things always have a way of working out in the end . The preparedness plan had to be completed, and adapted to Iceland's particular global situation. In the 21st century, the world has faced new epidemic threats, such as SARS, and old scourges such as the plague have resurfaced . One of the main findings on Ebola preparedness measures in the EU was that measures taken were based on past preparedness and experience of other epidemics, such as SARS and H1N1 . Further, key stakeholders within each country found their measures to have been adequate for dealing with a single case of Ebola, as was the case in Iceland. A preparedness plan for pandemic influenzae in Iceland was elaborated in 2006activated in response to the H1N1 epidemic in 2009and revised in 2016 .",
"Further, key stakeholders within each country found their measures to have been adequate for dealing with a single case of Ebola, as was the case in Iceland. A preparedness plan for pandemic influenzae in Iceland was elaborated in 2006activated in response to the H1N1 epidemic in 2009and revised in 2016 . During the elaboration of these plans, communication among the different levels of the healthcare system and supporting agencies, such as the DCPEM, had been clearly defined, and proved to be useful in the preparedness for Ebola. Further, as found important in preparedness activities for pandemic influenzae elsewhere , honesty, transparency in communication, and sharing of information from managers to front-line health professionals, was found to be critical. It gave a feeling of being involved, and mitigated the fear that is so frequently encountered during epidemics . Iceland was far away from the epicentre of the Ebola epidemic in West Africa.",
"It gave a feeling of being involved, and mitigated the fear that is so frequently encountered during epidemics . Iceland was far away from the epicentre of the Ebola epidemic in West Africa. Yet this case study shows that health professionals felt the strain of possibly having to treat one or more patients with EVD. Their situation stands in sharp contrast to the situation in the three worst affected West African countries that lacked staff, stuff, space, and systems to effectively address the challenge of EVD. Although Icelandic health professionals had trust in the national healthcare system, and in their own capacity, doubt and fear influenced the reflections on preparedness planning of both administrators and healthcare staff. References to national identity and the characteristic of an 'Icelandic approach' to handling challenges assisted participants in coming to terms with the experienced shortcomings of the preparedness plan, and underscored the pride in the ingenuity applied in the process.",
"Although Icelandic health professionals had trust in the national healthcare system, and in their own capacity, doubt and fear influenced the reflections on preparedness planning of both administrators and healthcare staff. References to national identity and the characteristic of an 'Icelandic approach' to handling challenges assisted participants in coming to terms with the experienced shortcomings of the preparedness plan, and underscored the pride in the ingenuity applied in the process. These references negotiate the role and character of the nation of Iceland, and its role in a globalized world, as both a small and isolated nation on one hand, and a central and capable one, on the other. The experienced ambiguity needs attention in a health system and among healthcare staff that have to act resolutely and unfailingly, should they be placed in charge of containing contamination. This study points to the necessity of repeatedly re-enacting, as realistically as possible, the likely scenarios of receiving and treating one or more patients infected with Ebola or other contagious global health threats as a routine matter. This would assist in the identification of overlooked 'soft periphery' concerns, and promote embodied preparedness among teams of health care staff on the frontline.",
"This study points to the necessity of repeatedly re-enacting, as realistically as possible, the likely scenarios of receiving and treating one or more patients infected with Ebola or other contagious global health threats as a routine matter. This would assist in the identification of overlooked 'soft periphery' concerns, and promote embodied preparedness among teams of health care staff on the frontline. Geir Gunnlaugsson conceptualized the study, and took part in all necessary steps towards its completion, such as analysis and interpretation of data, and writing the manuscript for submission. Íris Eva Hauksdóttir collected and analysed the data as part of a master thesis work conducted under the supervision of all three co-authors, revised the manuscript, and approved the final version. Ib Bygbjerg took part in the interpretation of data, revision of the manuscript, and approved the final version. Britt Pinkowski Tersbøl took part in designing interview tools and in the thematic analysis of interview data, interpretation, revision of the manuscript, and approved the final version.",
"Ib Bygbjerg took part in the interpretation of data, revision of the manuscript, and approved the final version. Britt Pinkowski Tersbøl took part in designing interview tools and in the thematic analysis of interview data, interpretation, revision of the manuscript, and approved the final version. Dr. Gunnlaugsson reports he was the Chief Medical Officer CMO for Iceland, Directorate of Health, in the period 2010-2014. Other authors report no conflict of interest. The study was reported to the Data Protection Authority and approved by the National Bioethics Committee in Iceland number VSI- . Subsequently, the study was approved by the University Hospital Ethical Committee on 4 February 2016 number LSH .",
"The study was reported to the Data Protection Authority and approved by the National Bioethics Committee in Iceland number VSI- . Subsequently, the study was approved by the University Hospital Ethical Committee on 4 February 2016 number LSH . Participants signed an informed consent form before taking part in the study. Not applicable. The manuscript builds on the work of Íris Eva Hauksdóttir towards a MSc in Global Health, Section of Global Health, Department of Public Health, Copenhagen University, Denmark."
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What is PPE? | Personal Protective Equipment | [
"Background: The Ebola epidemic in West Africa caused global fear and stirred up worldwide preparedness activities in countries sharing borders with those affected, and in geographically far-away countries such as Iceland. Objective: To describe and analyse Ebola preparedness activities within the Icelandic healthcare system, and to explore the perspectives and experiences of managers and frontline health workers. Methods: A qualitative case study, based on semi-structured interviews with 21 staff members in the national Ebola Treatment Team, Emergency Room at Landspitali University Hospital, and managers of the response team. Results: Contextual factors such as culture and demography influenced preparedness, and contributed to the positive state of mind of participants, and ingenuity in using available resources for preparedness. While participants believed they were ready to take on the task of Ebola, they also had doubts about the chances of Ebola ever reaching Iceland. Yet, factors such as fear of Ebola and the perceived stigma associated with caring for a potentially infected Ebola patient, influenced the preparation process and resulted in plans for specific precautions by staff to secure the safety of their families.",
"While participants believed they were ready to take on the task of Ebola, they also had doubts about the chances of Ebola ever reaching Iceland. Yet, factors such as fear of Ebola and the perceived stigma associated with caring for a potentially infected Ebola patient, influenced the preparation process and resulted in plans for specific precautions by staff to secure the safety of their families. There were also concerns about the teamwork and lack of commitment by some during training. Being a ‘tiny’ nation was seen as both an asset and a weakness in the preparation process. Honest information sharing and scenario-based training contributed to increased confidence amongst participants in the response plans. Conclusions: Communication and training were important for preparedness of health staff in Iceland, in order to receive, admit, and treat a patient suspected of having Ebola, while doubts prevailed on staff capacity to properly do so.",
"Honest information sharing and scenario-based training contributed to increased confidence amongst participants in the response plans. Conclusions: Communication and training were important for preparedness of health staff in Iceland, in order to receive, admit, and treat a patient suspected of having Ebola, while doubts prevailed on staff capacity to properly do so. For optimal preparedness, likely scenarios for future global security health threats need to be repeatedly enacted, and areas plagued by poverty and fragile healthcare systems require global support. Text: Global health; prevention and control; public policy; qualitative evaluation; emergency responders; communicable diseases; emerging; fear Background On 8 August 2014, the World Health Organization declared the Ebola epidemic in West Africa as a Public Health Emergency of International Concern PHEIC under the International Health Regulations IHR . All three of the worst affected countries were to address the emerging epidemic challenge without staff, stuff, space and systems . With the epidemic seemingly out of control, and a proportionately high number of doctors, nurses, and midwives succumbing to Ebola , there was a growing fear of transmission beyond the region.",
"All three of the worst affected countries were to address the emerging epidemic challenge without staff, stuff, space and systems . With the epidemic seemingly out of control, and a proportionately high number of doctors, nurses, and midwives succumbing to Ebola , there was a growing fear of transmission beyond the region. In breach of WHO recommendations and guidelines , flights were cancelled and cross-border movement curtailed . The epidemic caused public concern outside West Africa , as fear and racism found fertile ground , and in an effort to stop the international spread of the disease, all states were advised to be prepared to detect, investigate, and manage Ebola cases . Preparedness as part of disaster risk reduction is defined as 'the knowledge and capacities developed by governments, response and recovery organizations, communities and individuals to effectively anticipate, respond to, and recover from the impacts of likely, imminent or current disasters' . Yet, preparedness is also enveloped in and influenced by the socio-cultural dimension at the individual, organizational, and national levels, and measures to manage outbreaks are not always accepted or accommodated by the communities to which they are applied .",
"Preparedness as part of disaster risk reduction is defined as 'the knowledge and capacities developed by governments, response and recovery organizations, communities and individuals to effectively anticipate, respond to, and recover from the impacts of likely, imminent or current disasters' . Yet, preparedness is also enveloped in and influenced by the socio-cultural dimension at the individual, organizational, and national levels, and measures to manage outbreaks are not always accepted or accommodated by the communities to which they are applied . An analysis of eight European countries' preparedness plans since 2009 for countering a future influenza A H1N1 pandemic revealed that the way plans were framed varied considerably, and ' told us something about how the different countries want pandemics and preparedness to be understood by the public' . More research was encouraged into cultural and social structures in the respective countries. In Iceland, information about the Ebola epidemic in West Africa came from several sources. The Directorate of Health DH first reported on the epidemic on 8 April 2014 .",
"In Iceland, information about the Ebola epidemic in West Africa came from several sources. The Directorate of Health DH first reported on the epidemic on 8 April 2014 . In Icelandic media, the rapid progress of the Ebola epidemic in West Africa was increasingly highlighted, and exported Ebola cases to Spain, USA, and elsewhere, were widely covered. Fear of a global epidemic was rife, and in media and online discussions, doubts were raised about the Icelandic health system´s capacity to take care of a patient with Ebola , despite its ranking as one of the best in the world . On 11 August 2014, three days after WHO declared PHEIC because of Ebola, DH encouraged Icelandic citizens to avoid visits to the area, if possible, and reported that the national epidemic preparedness plan was being activated for Ebola . It was elaborated by a team that involved the Chief Epidemiologist at the DH, Landspitali University Hospital LSH , the Department of Civil Protection and Emergency Management DCPEM , and the seven Primary Healthcare Regional Organizations in the country at the time.",
"On 11 August 2014, three days after WHO declared PHEIC because of Ebola, DH encouraged Icelandic citizens to avoid visits to the area, if possible, and reported that the national epidemic preparedness plan was being activated for Ebola . It was elaborated by a team that involved the Chief Epidemiologist at the DH, Landspitali University Hospital LSH , the Department of Civil Protection and Emergency Management DCPEM , and the seven Primary Healthcare Regional Organizations in the country at the time. Key external partners were the European Centre for Disease Prevention and Control ECDC and WHO, in addition to Nordic collaborators in epidemic preparedness . At the same time, it was regarded as highly unlikely that Ebola Virus Disease EVD would spread in the country . Recognized scenarios included the possible appearance of an infected person in need of treatment, who could be either an Icelandic citizen who had visited or worked in one of the affected West African countries, or a person with signs of EVD on a trans-Atlantic flight in the navigation area controlled by Icelandic authorities . On 3 November 2014, the plan was put to the test when a foreign airline made a non-scheduled landing at Keflavík International Airport due to fear of EVD in one passenger from South Africa.",
"Recognized scenarios included the possible appearance of an infected person in need of treatment, who could be either an Icelandic citizen who had visited or worked in one of the affected West African countries, or a person with signs of EVD on a trans-Atlantic flight in the navigation area controlled by Icelandic authorities . On 3 November 2014, the plan was put to the test when a foreign airline made a non-scheduled landing at Keflavík International Airport due to fear of EVD in one passenger from South Africa. Parked in a closed-off area, a physician in full Personal Protective Equipment PPE entered the plane, but quickly ruled out Ebola . Irrespective of good or bad overall performance, health systems are tested in times of crisis, such as epidemics. Here, the aim is to describe and analyse the process of establishing preparedness plans for Ebola in Iceland, with a specific focus on the perspectives and experiences of managers and frontline health workers involved in the process. This study is part of a larger study on the impact that the global threat of the Ebola epidemic had in Iceland .",
"Here, the aim is to describe and analyse the process of establishing preparedness plans for Ebola in Iceland, with a specific focus on the perspectives and experiences of managers and frontline health workers involved in the process. This study is part of a larger study on the impact that the global threat of the Ebola epidemic had in Iceland . Qualitative case study methodology was applied, perceiving the preparedness planning and training process as the case with clear boundaries of the initiation, process, and wrap-up of preparedness planning and training. The study was conducted in April-May 2016, and the interviewed participants were administrators and frontline health professionals central to the case, so as to explore their perspectives and experiences concerning Ebola preparedness . Staff in managerial positions were contacted by one of the authors GG for permission to interview them based on their role in the preparedness plan. To identify potential interviewees in the Ebola Treatment Team ETT , the director of the team listed relevant email contacts.",
"Staff in managerial positions were contacted by one of the authors GG for permission to interview them based on their role in the preparedness plan. To identify potential interviewees in the Ebola Treatment Team ETT , the director of the team listed relevant email contacts. Those who responded positively were subsequently invited for an interview, conducted in Icelandic by one of the authors ÍEH , a physiotherapist. In case interviewees suggested other potential participants, they were invited through email to participate. A similar methodology was applied to identify participants from the Emergency Room ER . They were included in order to represent frontline health workers who worked in the only ER in Reykjavík, where persons exposed to EVD were most likely to first seek care in case of acute illness.",
"A similar methodology was applied to identify participants from the Emergency Room ER . They were included in order to represent frontline health workers who worked in the only ER in Reykjavík, where persons exposed to EVD were most likely to first seek care in case of acute illness. Three separate interview guides were developedone each for managers, ETT, and ER respectively see supplementary material . The interviews included open questions probing the role of their institution in preparedness, the experience of the training process, challenges encountered or expected, and any dilemmas that they may have experienced in relation to the preparedness plan. The recruitment of participants was concluded when saturation was reached. Each interview was recorded and took about 20 to 60 minutes; they were then transcribed and analysed using thematic analysis.",
"The recruitment of participants was concluded when saturation was reached. Each interview was recorded and took about 20 to 60 minutes; they were then transcribed and analysed using thematic analysis. The data material was read through repeatedly, sorted, and categorized, based on the participants' priorities in the representation of their views. From this exercise, three broad themes were inductively identified that corresponded to critical perspectives introduced by the participants. Permission to conduct the study was granted by Iceland's National Bioethics Committee VSN- and Landspitali University Hospital LSH 13-16, 4 February 2016 . Reporting on the results was guided by the COREC guidelines ; however, to ensure anonymity of the respondents within the small community of staff who took part in the preparedness activities, participant information is not associated to quotations.",
"Permission to conduct the study was granted by Iceland's National Bioethics Committee VSN- and Landspitali University Hospital LSH 13-16, 4 February 2016 . Reporting on the results was guided by the COREC guidelines ; however, to ensure anonymity of the respondents within the small community of staff who took part in the preparedness activities, participant information is not associated to quotations. The Icelandic Ebola Preparedness Plan included the establishment of an ETT within LSH , and the preparatory activities engaged more than two hundred staff across all of its departments. The ETT consisted of about 50 healthcare professionals who had volunteered to participate, including 11 doctors and 28 nurses, a few laboratory technicians, radiologists, and auxiliary nurses. They attended special training sessions focused on protocols for admission and treatment of a patient with EVD, the donning/doffing of PPE, and personal protective measures during patient care. A new provisory unit was designed to be set up on the ground floor to minimize the risk of infection spreading to other units within the hospital, with two rooms specifically identified for the care of a patient with EVD .",
"They attended special training sessions focused on protocols for admission and treatment of a patient with EVD, the donning/doffing of PPE, and personal protective measures during patient care. A new provisory unit was designed to be set up on the ground floor to minimize the risk of infection spreading to other units within the hospital, with two rooms specifically identified for the care of a patient with EVD . Managers' accounts of this period elaborated the complexity of preparedness planning in terms of the involved institutions, actors, procedures and requirement of the plan. One manager concluded: You get no discount. You can never go the shorter way. There was always something that surprised you.",
"One manager concluded: You get no discount. You can never go the shorter way. There was always something that surprised you. We thought this was a lot like a three headed monster, so when you chopped off one of its heads, three other emerged, every solution was followed by more problems. The health professionals who volunteered to join ETT did so for different reasons. Ebola preparedness was 'a job that had to be done', and 'someone had to do it'.",
"The health professionals who volunteered to join ETT did so for different reasons. Ebola preparedness was 'a job that had to be done', and 'someone had to do it'. Some referred to ethical or professional obligations: This is just a part of being a nurse, to encounter situations that can be dangerous to you or someone else, but you have made this decision and you deal with it. Some connected their decision to their 'action gene' or 'addiction to taking risks', while others said they had already raised their kids and had years of experience, including work with other epidemics, such as HIV. Yet, the practice of volunteering in the preparation was questioned. One participant said: We learned that we could not rely on volunteers … when you work in an infectious disease department you cannot choose what infections you want to work with.",
"Yet, the practice of volunteering in the preparation was questioned. One participant said: We learned that we could not rely on volunteers … when you work in an infectious disease department you cannot choose what infections you want to work with. ER staff indicated that for them working in the ER was enough of a risk to take, no reason to expose oneself even more by joining the ETT, and appreciated that others had volunteered. All participants noted that co-operation and communication had generally functioned well during the preparedness planning, with information flowing both ways. Short communication lines within the healthcare system were perceived as both a strength and a weakness; a strength, insofar as people knew each other, but a weakness because of the uneven burden of workload. Staff of the ETT and in the ER felt they had been well-informed, and that openness and honesty had characterized the planning and diminished their initial fear.",
"Short communication lines within the healthcare system were perceived as both a strength and a weakness; a strength, insofar as people knew each other, but a weakness because of the uneven burden of workload. Staff of the ETT and in the ER felt they had been well-informed, and that openness and honesty had characterized the planning and diminished their initial fear. Those in managerial positions had listened and taken their opinions into consideration. One said: They were honest, no one was hiding anything, everything was on the table, no one tried to make things more appealing and say that everything would be OK, they just told us about things as they were. Both management and participants from the ETT and ER expressed their ambiguity in terms of trust, doubt, and fear. Participants conveyed trust in the health system and their own role as health professionals, while at the same time admitting to facing formidable challenges during the elaboration of the preparedness plan.",
"Both management and participants from the ETT and ER expressed their ambiguity in terms of trust, doubt, and fear. Participants conveyed trust in the health system and their own role as health professionals, while at the same time admitting to facing formidable challenges during the elaboration of the preparedness plan. Facilities for isolation and treatment of patients with Ebola were less than perfect: We assessed how we could use the department … and change it in just a few hours into some kind of an isolation unit that we could possibly use. Some compared this short-term isolation facility to a 'camping site', as the facilities were too provisional and not comparable to those found elsewhere. There was also doubt about how many Ebola patients LSH would be able to care for: 'Maybe one or two patients, barely more'. Respondents believed that the training and education of the members of the ETT and ER had been satisfactory.",
"There was also doubt about how many Ebola patients LSH would be able to care for: 'Maybe one or two patients, barely more'. Respondents believed that the training and education of the members of the ETT and ER had been satisfactory. They felt that it had been proportionate to the risk, while some were concerned about the lack of staff. Nonetheless, there were contradictions on the division of labour among the professionals, exemplified by different ideas on how to proceed if a patient suspected of having an EVD came in an ambulance to the LSH for treatment. Almost all participants stated that they were ready to do their part in the Ebola response, or 'as ready as we could be'. There were diverse opinions on what it meant to be ready: to treat one confirmed case of Ebola, one suspected case, or more EVD patients?",
"Almost all participants stated that they were ready to do their part in the Ebola response, or 'as ready as we could be'. There were diverse opinions on what it meant to be ready: to treat one confirmed case of Ebola, one suspected case, or more EVD patients? When asked if Ebola was a real threat to the country, participants usually referred to how easy it was to travel the globe: 'Yeah, why not, the world is getting smaller'. Although Ebola was thought of as a real danger by many, some participants expressed difficulty in taking their training seriously, doubting that Ebola would ever reach Iceland. One respondent said: People were dedicated in the beginning, but when the news appeared that Ebola was receding, that diminished, and I never felt like this formally ended. Participants described their relief that nothing really happened, while emphasizing the need to experience a real situation to evaluate the preparedness efforts.",
"One respondent said: People were dedicated in the beginning, but when the news appeared that Ebola was receding, that diminished, and I never felt like this formally ended. Participants described their relief that nothing really happened, while emphasizing the need to experience a real situation to evaluate the preparedness efforts. One participant said that 'a little bit more seriousness would have been needed in the PPE practices'. It was taken as a manifestation of fear that some of the staff in the communicable disease department of the LSH refused to take part in the ETT. When describing their fears, ETT members frequently connected it to their working conditions. Many of them were afraid that they would not get the best PPE, others that they would not do the donning/doffing correctly and, lastly, they were worried about work performance while in the PPE.",
"When describing their fears, ETT members frequently connected it to their working conditions. Many of them were afraid that they would not get the best PPE, others that they would not do the donning/doffing correctly and, lastly, they were worried about work performance while in the PPE. One participant said: What bothered most of us was how uncomfortable the PPE was and I think that made people nervous: \"How will I manage working in this for hours?\" Another described the donning/doffing process like a 'complicated ballroom dance'. Moreover, participants were afraid of 'unknown territories', that is, they did not know the hospital ward, they were supposed to work in, and some team members had no recent experience of clinical work. One participant said: I didn't think these non-clinical people belonged in the team, because this is a very clinical environment in addition to having to be in this costume PPE with the risk of becoming infected by mistake.",
"Moreover, participants were afraid of 'unknown territories', that is, they did not know the hospital ward, they were supposed to work in, and some team members had no recent experience of clinical work. One participant said: I didn't think these non-clinical people belonged in the team, because this is a very clinical environment in addition to having to be in this costume PPE with the risk of becoming infected by mistake. Those with non-clinical background were, however, aware of their limitations: I realized that I would not be the one in the front, I would not be managing patients directly. The importance ascribed to teamwork was evident in relation to fear. Participants described fear of working with people they had not worked with before: The weakest link in the preparation was that even though I knew their faces, I had never worked with them. Another issue was no-show by some team members in training sessions or in lectures: This is team-work, one does this and the other one does this, we help each other.",
"Participants described fear of working with people they had not worked with before: The weakest link in the preparation was that even though I knew their faces, I had never worked with them. Another issue was no-show by some team members in training sessions or in lectures: This is team-work, one does this and the other one does this, we help each other. Then you don't want to be working with someone who didn't show up. There were a lot of doctors who just dropped in, dropped out, and then dropped in again. I asked myself: Are these individuals … ready to take this on? Participants in the ETT mentioned the precautions they took or intended to take to cope with their feelings of fear, should Ebola emerge in Iceland.",
"I asked myself: Are these individuals … ready to take this on? Participants in the ETT mentioned the precautions they took or intended to take to cope with their feelings of fear, should Ebola emerge in Iceland. A major precaution was planning to avoid contact with the family while working with Ebola patients. One participant said: 'You thought … about your children at school … parents in the neighbourhood …' if they knew s he was working with an Ebola patient. For them, it was important they would have access to special accommodation in case of clinical EVD work 'so I wouldn't be exposing anyone or creating hysteria'. ETT members mentioned the extra insurance offered as a prerequisite for taking part in the team.",
"For them, it was important they would have access to special accommodation in case of clinical EVD work 'so I wouldn't be exposing anyone or creating hysteria'. ETT members mentioned the extra insurance offered as a prerequisite for taking part in the team. 'The normal insurance for LHS staff would not cover everything if we were to become sick or even lose our lives.' Amongst ER staff, the matter of insurance did seem to be less of an issue compared to the ETT. One respondent said: 'You are used to being at risk by many disease threats'. Furthermore, the issue of higher salaries and risk commission came up in the interviews, but overall did not matter as much to the participants as the insurance, or assurance of accommodation in case of need.",
"One respondent said: 'You are used to being at risk by many disease threats'. Furthermore, the issue of higher salaries and risk commission came up in the interviews, but overall did not matter as much to the participants as the insurance, or assurance of accommodation in case of need. Characteristics associated with Iceland and the Icelandic people were referred to repeatedly by participants. The concept 'Tiny Iceland' was often mentioned and emerged with positive and negative connotations. 'Tiny Iceland' referred to the size of the country and population and its perceived capability to still 'get the job done'. even though compromises had to be made.",
"'Tiny Iceland' referred to the size of the country and population and its perceived capability to still 'get the job done'. even though compromises had to be made. Comparing how Iceland handled its responsibilities differently from other countries of a larger size was often brought up, both with pride in Iceland as a strong independent nation, and with insecurities about its capacity in comparison to other countries. It was pointed out that since the preparedness process was in the hands of a few people, everyone knew their role. As one administrator said: This little hospital system, as complicated as it might seem every day, gives you the chance to just pick up the phone and call the one in charge. Being a small population presents challenges regarding resources, infrastructure, and specialized medical training to comply with standards of international actors.",
"As one administrator said: This little hospital system, as complicated as it might seem every day, gives you the chance to just pick up the phone and call the one in charge. Being a small population presents challenges regarding resources, infrastructure, and specialized medical training to comply with standards of international actors. Notions of Icelanders as resilient in spite of shortcomings were common; referring to the experience of preparedness planning and training, one health staff said: It was very much the Icelandic way, we'll manage, we'll work it out, and there was so much ingenuity. This notion of a particular Icelandic approach to coping, in spite of shortcomings, was also detected more generally, as in the statement: Would it have worked? Yes, it would have worked. Would it have been optimal?",
"Yes, it would have worked. Would it have been optimal? We cannot say, it would have been optimal; we can say, it would have been sufficient. In contrast to this, there were concerns about whether Icelandic aid workers falling ill in Ebolaaffected countries should be transferred to Iceland or to hospitals in other Nordic countries with better isolation units. Some of the participants trusted that patients with EVD would not be transferred to Iceland. One participant stated: You heard that Norwegians were criticized for transferring their aid worker from Africa to Norway. We don't know what would have happened if they would have transferred an Icelander into the country.",
"One participant stated: You heard that Norwegians were criticized for transferring their aid worker from Africa to Norway. We don't know what would have happened if they would have transferred an Icelander into the country. We don't have good enough isolation unitsyou are not supposed to send patients to a hospital that is less than 100%. I thought there was assurance in that. During the devastating Ebola epidemic in West Africa that spread to neighbouring sub-Saharan countries, North America, and Europe , preparedness plans were widely elaborated and later evaluated. Evaluations have, for example, been conducted in 11 African countries close to the epidemic , in the EU region , and the US .",
"During the devastating Ebola epidemic in West Africa that spread to neighbouring sub-Saharan countries, North America, and Europe , preparedness plans were widely elaborated and later evaluated. Evaluations have, for example, been conducted in 11 African countries close to the epidemic , in the EU region , and the US . Here we present data from a qualitative case study on the process, and experiences with establishing a preparedness plan for Ebola in Iceland in 2014. Interviews with staff who were engaged, either as administrators or frontline healthcare workers, alert us to the manner in which geographic, demographic, cultural, and organizational characteristics shaped the response. The results show that the process of establishing and training for preparedness was permeated by ambiguities of pride and pragmatism, trust, doubts, and fear. 'Getting the job done' theme 1 refers to the multitude of tasks and considerations that surrounds and feeds into the preparedness plan itself and are necessary for successful planning and implementation.",
"The results show that the process of establishing and training for preparedness was permeated by ambiguities of pride and pragmatism, trust, doubts, and fear. 'Getting the job done' theme 1 refers to the multitude of tasks and considerations that surrounds and feeds into the preparedness plan itself and are necessary for successful planning and implementation. Using the metaphors of 'hard core' and 'soft periphery', Langley and Denis emphasize the importance of relatively 'peripheral' concerns and processes for planning and implementation of new interventions. The hard core represents the actual intervention or goal, e.g. implementation of a preparedness plan. The soft periphery refers to all the contextually important networking, negotiations, and agreements necessary to deliver the hard core.",
"implementation of a preparedness plan. The soft periphery refers to all the contextually important networking, negotiations, and agreements necessary to deliver the hard core. If the soft periphery is neglected, it will cause multiple challenges in the implementation process, and the benefit of the hard core, the intervention itself, may not transpire as anticipated. Due attention to the soft periphery may, however, considerably promote the delivery of an innovation, and secure support from important stakeholders. In our data, one manager speaks of the preparedness process as dealing with a three-headed monster where every solution was followed by new problems. The data indicate that the process of dealing with 'the three headed monster' was given due attention as a means to successfully develop Iceland's preparedness plan.",
"In our data, one manager speaks of the preparedness process as dealing with a three-headed monster where every solution was followed by new problems. The data indicate that the process of dealing with 'the three headed monster' was given due attention as a means to successfully develop Iceland's preparedness plan. Comprehensive consultations and the involvement of many associated institutions were mentioned. Still ambiguity remained with some staff in terms of division of responsibilities and taskse.g. when transporting a patient potentially infected with Ebola from the airport to the hospital, and other such activities. During epidemics, rumours, gossip, and unreliable information on the news and social media spread rapidly, resulting in so-called 'infodemics' .",
"when transporting a patient potentially infected with Ebola from the airport to the hospital, and other such activities. During epidemics, rumours, gossip, and unreliable information on the news and social media spread rapidly, resulting in so-called 'infodemics' . The West African Ebola epidemic was covered widely by media , and the fear of Ebola reached every corner of the world, exemplified by travel bans from affected countries, and trade barriers , in contrast to the ongoing epidemic in the Democratic Republic of Congo . In our second theme, trust, doubt, and fear of health workers were represented. Although all intentions were good, concerns remained about the suitability and safety of the isolation ward, the PPE, and other tools, as well as adequate engagement of colleagues who might potentially work alongside them, in case an Ebola patient came to Iceland. The foreignness of putting on, removing, and working from within a PPE and the trustworthiness of available PPE were mentioned.",
"Although all intentions were good, concerns remained about the suitability and safety of the isolation ward, the PPE, and other tools, as well as adequate engagement of colleagues who might potentially work alongside them, in case an Ebola patient came to Iceland. The foreignness of putting on, removing, and working from within a PPE and the trustworthiness of available PPE were mentioned. In preparedness efforts in other countries, scarcity of resources in relation to manpower demand and problems with training and protocols involving PPE were common challenges . Similar problems were encountered in Iceland. Provisory treatment facility had to be designed, called 'camping site' by some, in contrast to facilities found elsewhere . Further, the ETT was established based on voluntary recruitment rather than on the staff's assigned roles within the healthcare system, a procedure that was deemed less than optimal.",
"Provisory treatment facility had to be designed, called 'camping site' by some, in contrast to facilities found elsewhere . Further, the ETT was established based on voluntary recruitment rather than on the staff's assigned roles within the healthcare system, a procedure that was deemed less than optimal. The members of the ETT pointed out that they had never worked together as a team under circumstances that demanded strict adherence to infectious control procedures. This eroded trust, compounded by the laissez-faire attitude of some of its members during the preparation exercises, possibly due to other competing tasks in a busy hospital and insufficient resources that hampered full participation . Further, it was a constraint that simulation exercises were not an option, found to be an important element in preparation for epidemics . This might have resulted in less than optimal staff protection for those who would have been in direct contact with an infected patient, as reported during the SARS epidemic in Canada .",
"Further, it was a constraint that simulation exercises were not an option, found to be an important element in preparation for epidemics . This might have resulted in less than optimal staff protection for those who would have been in direct contact with an infected patient, as reported during the SARS epidemic in Canada . Anthropological work on emergency preparedness emphasizes the connectedness between health professionals, technological devices, and knowledge as a prerequisite for successful preparedness. Wolf and Hall present preparedness efforts as a form of governance that involves human bodies those of health professionals , clinical architectures e.g. isolation wards , and technical artefacts gloves, protective suits, disinfectants, etc. .",
"isolation wards , and technical artefacts gloves, protective suits, disinfectants, etc. . During preparedness training and implementation, 'nursing bodies are transformed into instruments of preparedness', and become part of infrastructural arrangements. Health professionals are, here, both vulnerable and powerful tools in the management of contamination. The authors argue that successful planning, training, and implementation of a preparedness plan require such intrinsic connectedness. In the case of Ebola preparedness in Iceland, health professionals draw our attention to dilemmas of connectedness, and their assessment of the fact that these shortcomings might hamper the mobilization of 'preparedness within the human body'that is, the embodied experience, routine, and tacit knowledge which Wolf and Hall state are key to successful implementation.",
"The authors argue that successful planning, training, and implementation of a preparedness plan require such intrinsic connectedness. In the case of Ebola preparedness in Iceland, health professionals draw our attention to dilemmas of connectedness, and their assessment of the fact that these shortcomings might hamper the mobilization of 'preparedness within the human body'that is, the embodied experience, routine, and tacit knowledge which Wolf and Hall state are key to successful implementation. Repeated enactment of receiving and treating a patient with Ebola within experienced and trustful teams would probably enhance such embodiment, provided that there is justified trust in the involved technology. In addition, repetition would also strengthen the 'soft periphery' of preparedness, and divisions of responsibilities would be clearer manifested. In the third theme, we observe how notions of the 'Icelandic way' help participants make sense of ambiguities about Ebola preparedness. Loftsdóttir explored how people negotiated the imagination of the local and the global during the 2008 economic crisis in Iceland .",
"In the third theme, we observe how notions of the 'Icelandic way' help participants make sense of ambiguities about Ebola preparedness. Loftsdóttir explored how people negotiated the imagination of the local and the global during the 2008 economic crisis in Iceland . Notions of the intrinsic character of Iceland, and of being Icelandic, serve to underscore certain points and explain positive and negative experiences with the preparedness plan. Iceland is far away from the continents, but still connected through global needs for policy, risk of contamination, and dependency in terms of collaboration, in emergencies emerging from elsewhere. In our study, participants highlighted the importance of believing in oneself and the 'Icelandic way of doing things,' summed up in the paraphrase 'þetta reddast' things always have a way of working out in the end . The preparedness plan had to be completed, and adapted to Iceland's particular global situation.",
"In our study, participants highlighted the importance of believing in oneself and the 'Icelandic way of doing things,' summed up in the paraphrase 'þetta reddast' things always have a way of working out in the end . The preparedness plan had to be completed, and adapted to Iceland's particular global situation. In the 21st century, the world has faced new epidemic threats, such as SARS, and old scourges such as the plague have resurfaced . One of the main findings on Ebola preparedness measures in the EU was that measures taken were based on past preparedness and experience of other epidemics, such as SARS and H1N1 . Further, key stakeholders within each country found their measures to have been adequate for dealing with a single case of Ebola, as was the case in Iceland. A preparedness plan for pandemic influenzae in Iceland was elaborated in 2006activated in response to the H1N1 epidemic in 2009and revised in 2016 .",
"Further, key stakeholders within each country found their measures to have been adequate for dealing with a single case of Ebola, as was the case in Iceland. A preparedness plan for pandemic influenzae in Iceland was elaborated in 2006activated in response to the H1N1 epidemic in 2009and revised in 2016 . During the elaboration of these plans, communication among the different levels of the healthcare system and supporting agencies, such as the DCPEM, had been clearly defined, and proved to be useful in the preparedness for Ebola. Further, as found important in preparedness activities for pandemic influenzae elsewhere , honesty, transparency in communication, and sharing of information from managers to front-line health professionals, was found to be critical. It gave a feeling of being involved, and mitigated the fear that is so frequently encountered during epidemics . Iceland was far away from the epicentre of the Ebola epidemic in West Africa.",
"It gave a feeling of being involved, and mitigated the fear that is so frequently encountered during epidemics . Iceland was far away from the epicentre of the Ebola epidemic in West Africa. Yet this case study shows that health professionals felt the strain of possibly having to treat one or more patients with EVD. Their situation stands in sharp contrast to the situation in the three worst affected West African countries that lacked staff, stuff, space, and systems to effectively address the challenge of EVD. Although Icelandic health professionals had trust in the national healthcare system, and in their own capacity, doubt and fear influenced the reflections on preparedness planning of both administrators and healthcare staff. References to national identity and the characteristic of an 'Icelandic approach' to handling challenges assisted participants in coming to terms with the experienced shortcomings of the preparedness plan, and underscored the pride in the ingenuity applied in the process.",
"Although Icelandic health professionals had trust in the national healthcare system, and in their own capacity, doubt and fear influenced the reflections on preparedness planning of both administrators and healthcare staff. References to national identity and the characteristic of an 'Icelandic approach' to handling challenges assisted participants in coming to terms with the experienced shortcomings of the preparedness plan, and underscored the pride in the ingenuity applied in the process. These references negotiate the role and character of the nation of Iceland, and its role in a globalized world, as both a small and isolated nation on one hand, and a central and capable one, on the other. The experienced ambiguity needs attention in a health system and among healthcare staff that have to act resolutely and unfailingly, should they be placed in charge of containing contamination. This study points to the necessity of repeatedly re-enacting, as realistically as possible, the likely scenarios of receiving and treating one or more patients infected with Ebola or other contagious global health threats as a routine matter. This would assist in the identification of overlooked 'soft periphery' concerns, and promote embodied preparedness among teams of health care staff on the frontline.",
"This study points to the necessity of repeatedly re-enacting, as realistically as possible, the likely scenarios of receiving and treating one or more patients infected with Ebola or other contagious global health threats as a routine matter. This would assist in the identification of overlooked 'soft periphery' concerns, and promote embodied preparedness among teams of health care staff on the frontline. Geir Gunnlaugsson conceptualized the study, and took part in all necessary steps towards its completion, such as analysis and interpretation of data, and writing the manuscript for submission. Íris Eva Hauksdóttir collected and analysed the data as part of a master thesis work conducted under the supervision of all three co-authors, revised the manuscript, and approved the final version. Ib Bygbjerg took part in the interpretation of data, revision of the manuscript, and approved the final version. Britt Pinkowski Tersbøl took part in designing interview tools and in the thematic analysis of interview data, interpretation, revision of the manuscript, and approved the final version.",
"Ib Bygbjerg took part in the interpretation of data, revision of the manuscript, and approved the final version. Britt Pinkowski Tersbøl took part in designing interview tools and in the thematic analysis of interview data, interpretation, revision of the manuscript, and approved the final version. Dr. Gunnlaugsson reports he was the Chief Medical Officer CMO for Iceland, Directorate of Health, in the period 2010-2014. Other authors report no conflict of interest. The study was reported to the Data Protection Authority and approved by the National Bioethics Committee in Iceland number VSI- . Subsequently, the study was approved by the University Hospital Ethical Committee on 4 February 2016 number LSH .",
"The study was reported to the Data Protection Authority and approved by the National Bioethics Committee in Iceland number VSI- . Subsequently, the study was approved by the University Hospital Ethical Committee on 4 February 2016 number LSH . Participants signed an informed consent form before taking part in the study. Not applicable. The manuscript builds on the work of Íris Eva Hauksdóttir towards a MSc in Global Health, Section of Global Health, Department of Public Health, Copenhagen University, Denmark."
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Where did the 2014 Ebola epidemic in West Africa spread to? | neighbouring sub-Saharan countries, North America, and Europe | [
"Background: The Ebola epidemic in West Africa caused global fear and stirred up worldwide preparedness activities in countries sharing borders with those affected, and in geographically far-away countries such as Iceland. Objective: To describe and analyse Ebola preparedness activities within the Icelandic healthcare system, and to explore the perspectives and experiences of managers and frontline health workers. Methods: A qualitative case study, based on semi-structured interviews with 21 staff members in the national Ebola Treatment Team, Emergency Room at Landspitali University Hospital, and managers of the response team. Results: Contextual factors such as culture and demography influenced preparedness, and contributed to the positive state of mind of participants, and ingenuity in using available resources for preparedness. While participants believed they were ready to take on the task of Ebola, they also had doubts about the chances of Ebola ever reaching Iceland. Yet, factors such as fear of Ebola and the perceived stigma associated with caring for a potentially infected Ebola patient, influenced the preparation process and resulted in plans for specific precautions by staff to secure the safety of their families.",
"While participants believed they were ready to take on the task of Ebola, they also had doubts about the chances of Ebola ever reaching Iceland. Yet, factors such as fear of Ebola and the perceived stigma associated with caring for a potentially infected Ebola patient, influenced the preparation process and resulted in plans for specific precautions by staff to secure the safety of their families. There were also concerns about the teamwork and lack of commitment by some during training. Being a ‘tiny’ nation was seen as both an asset and a weakness in the preparation process. Honest information sharing and scenario-based training contributed to increased confidence amongst participants in the response plans. Conclusions: Communication and training were important for preparedness of health staff in Iceland, in order to receive, admit, and treat a patient suspected of having Ebola, while doubts prevailed on staff capacity to properly do so.",
"Honest information sharing and scenario-based training contributed to increased confidence amongst participants in the response plans. Conclusions: Communication and training were important for preparedness of health staff in Iceland, in order to receive, admit, and treat a patient suspected of having Ebola, while doubts prevailed on staff capacity to properly do so. For optimal preparedness, likely scenarios for future global security health threats need to be repeatedly enacted, and areas plagued by poverty and fragile healthcare systems require global support. Text: Global health; prevention and control; public policy; qualitative evaluation; emergency responders; communicable diseases; emerging; fear Background On 8 August 2014, the World Health Organization declared the Ebola epidemic in West Africa as a Public Health Emergency of International Concern PHEIC under the International Health Regulations IHR . All three of the worst affected countries were to address the emerging epidemic challenge without staff, stuff, space and systems . With the epidemic seemingly out of control, and a proportionately high number of doctors, nurses, and midwives succumbing to Ebola , there was a growing fear of transmission beyond the region.",
"All three of the worst affected countries were to address the emerging epidemic challenge without staff, stuff, space and systems . With the epidemic seemingly out of control, and a proportionately high number of doctors, nurses, and midwives succumbing to Ebola , there was a growing fear of transmission beyond the region. In breach of WHO recommendations and guidelines , flights were cancelled and cross-border movement curtailed . The epidemic caused public concern outside West Africa , as fear and racism found fertile ground , and in an effort to stop the international spread of the disease, all states were advised to be prepared to detect, investigate, and manage Ebola cases . Preparedness as part of disaster risk reduction is defined as 'the knowledge and capacities developed by governments, response and recovery organizations, communities and individuals to effectively anticipate, respond to, and recover from the impacts of likely, imminent or current disasters' . Yet, preparedness is also enveloped in and influenced by the socio-cultural dimension at the individual, organizational, and national levels, and measures to manage outbreaks are not always accepted or accommodated by the communities to which they are applied .",
"Preparedness as part of disaster risk reduction is defined as 'the knowledge and capacities developed by governments, response and recovery organizations, communities and individuals to effectively anticipate, respond to, and recover from the impacts of likely, imminent or current disasters' . Yet, preparedness is also enveloped in and influenced by the socio-cultural dimension at the individual, organizational, and national levels, and measures to manage outbreaks are not always accepted or accommodated by the communities to which they are applied . An analysis of eight European countries' preparedness plans since 2009 for countering a future influenza A H1N1 pandemic revealed that the way plans were framed varied considerably, and ' told us something about how the different countries want pandemics and preparedness to be understood by the public' . More research was encouraged into cultural and social structures in the respective countries. In Iceland, information about the Ebola epidemic in West Africa came from several sources. The Directorate of Health DH first reported on the epidemic on 8 April 2014 .",
"In Iceland, information about the Ebola epidemic in West Africa came from several sources. The Directorate of Health DH first reported on the epidemic on 8 April 2014 . In Icelandic media, the rapid progress of the Ebola epidemic in West Africa was increasingly highlighted, and exported Ebola cases to Spain, USA, and elsewhere, were widely covered. Fear of a global epidemic was rife, and in media and online discussions, doubts were raised about the Icelandic health system´s capacity to take care of a patient with Ebola , despite its ranking as one of the best in the world . On 11 August 2014, three days after WHO declared PHEIC because of Ebola, DH encouraged Icelandic citizens to avoid visits to the area, if possible, and reported that the national epidemic preparedness plan was being activated for Ebola . It was elaborated by a team that involved the Chief Epidemiologist at the DH, Landspitali University Hospital LSH , the Department of Civil Protection and Emergency Management DCPEM , and the seven Primary Healthcare Regional Organizations in the country at the time.",
"On 11 August 2014, three days after WHO declared PHEIC because of Ebola, DH encouraged Icelandic citizens to avoid visits to the area, if possible, and reported that the national epidemic preparedness plan was being activated for Ebola . It was elaborated by a team that involved the Chief Epidemiologist at the DH, Landspitali University Hospital LSH , the Department of Civil Protection and Emergency Management DCPEM , and the seven Primary Healthcare Regional Organizations in the country at the time. Key external partners were the European Centre for Disease Prevention and Control ECDC and WHO, in addition to Nordic collaborators in epidemic preparedness . At the same time, it was regarded as highly unlikely that Ebola Virus Disease EVD would spread in the country . Recognized scenarios included the possible appearance of an infected person in need of treatment, who could be either an Icelandic citizen who had visited or worked in one of the affected West African countries, or a person with signs of EVD on a trans-Atlantic flight in the navigation area controlled by Icelandic authorities . On 3 November 2014, the plan was put to the test when a foreign airline made a non-scheduled landing at Keflavík International Airport due to fear of EVD in one passenger from South Africa.",
"Recognized scenarios included the possible appearance of an infected person in need of treatment, who could be either an Icelandic citizen who had visited or worked in one of the affected West African countries, or a person with signs of EVD on a trans-Atlantic flight in the navigation area controlled by Icelandic authorities . On 3 November 2014, the plan was put to the test when a foreign airline made a non-scheduled landing at Keflavík International Airport due to fear of EVD in one passenger from South Africa. Parked in a closed-off area, a physician in full Personal Protective Equipment PPE entered the plane, but quickly ruled out Ebola . Irrespective of good or bad overall performance, health systems are tested in times of crisis, such as epidemics. Here, the aim is to describe and analyse the process of establishing preparedness plans for Ebola in Iceland, with a specific focus on the perspectives and experiences of managers and frontline health workers involved in the process. This study is part of a larger study on the impact that the global threat of the Ebola epidemic had in Iceland .",
"Here, the aim is to describe and analyse the process of establishing preparedness plans for Ebola in Iceland, with a specific focus on the perspectives and experiences of managers and frontline health workers involved in the process. This study is part of a larger study on the impact that the global threat of the Ebola epidemic had in Iceland . Qualitative case study methodology was applied, perceiving the preparedness planning and training process as the case with clear boundaries of the initiation, process, and wrap-up of preparedness planning and training. The study was conducted in April-May 2016, and the interviewed participants were administrators and frontline health professionals central to the case, so as to explore their perspectives and experiences concerning Ebola preparedness . Staff in managerial positions were contacted by one of the authors GG for permission to interview them based on their role in the preparedness plan. To identify potential interviewees in the Ebola Treatment Team ETT , the director of the team listed relevant email contacts.",
"Staff in managerial positions were contacted by one of the authors GG for permission to interview them based on their role in the preparedness plan. To identify potential interviewees in the Ebola Treatment Team ETT , the director of the team listed relevant email contacts. Those who responded positively were subsequently invited for an interview, conducted in Icelandic by one of the authors ÍEH , a physiotherapist. In case interviewees suggested other potential participants, they were invited through email to participate. A similar methodology was applied to identify participants from the Emergency Room ER . They were included in order to represent frontline health workers who worked in the only ER in Reykjavík, where persons exposed to EVD were most likely to first seek care in case of acute illness.",
"A similar methodology was applied to identify participants from the Emergency Room ER . They were included in order to represent frontline health workers who worked in the only ER in Reykjavík, where persons exposed to EVD were most likely to first seek care in case of acute illness. Three separate interview guides were developedone each for managers, ETT, and ER respectively see supplementary material . The interviews included open questions probing the role of their institution in preparedness, the experience of the training process, challenges encountered or expected, and any dilemmas that they may have experienced in relation to the preparedness plan. The recruitment of participants was concluded when saturation was reached. Each interview was recorded and took about 20 to 60 minutes; they were then transcribed and analysed using thematic analysis.",
"The recruitment of participants was concluded when saturation was reached. Each interview was recorded and took about 20 to 60 minutes; they were then transcribed and analysed using thematic analysis. The data material was read through repeatedly, sorted, and categorized, based on the participants' priorities in the representation of their views. From this exercise, three broad themes were inductively identified that corresponded to critical perspectives introduced by the participants. Permission to conduct the study was granted by Iceland's National Bioethics Committee VSN- and Landspitali University Hospital LSH 13-16, 4 February 2016 . Reporting on the results was guided by the COREC guidelines ; however, to ensure anonymity of the respondents within the small community of staff who took part in the preparedness activities, participant information is not associated to quotations.",
"Permission to conduct the study was granted by Iceland's National Bioethics Committee VSN- and Landspitali University Hospital LSH 13-16, 4 February 2016 . Reporting on the results was guided by the COREC guidelines ; however, to ensure anonymity of the respondents within the small community of staff who took part in the preparedness activities, participant information is not associated to quotations. The Icelandic Ebola Preparedness Plan included the establishment of an ETT within LSH , and the preparatory activities engaged more than two hundred staff across all of its departments. The ETT consisted of about 50 healthcare professionals who had volunteered to participate, including 11 doctors and 28 nurses, a few laboratory technicians, radiologists, and auxiliary nurses. They attended special training sessions focused on protocols for admission and treatment of a patient with EVD, the donning/doffing of PPE, and personal protective measures during patient care. A new provisory unit was designed to be set up on the ground floor to minimize the risk of infection spreading to other units within the hospital, with two rooms specifically identified for the care of a patient with EVD .",
"They attended special training sessions focused on protocols for admission and treatment of a patient with EVD, the donning/doffing of PPE, and personal protective measures during patient care. A new provisory unit was designed to be set up on the ground floor to minimize the risk of infection spreading to other units within the hospital, with two rooms specifically identified for the care of a patient with EVD . Managers' accounts of this period elaborated the complexity of preparedness planning in terms of the involved institutions, actors, procedures and requirement of the plan. One manager concluded: You get no discount. You can never go the shorter way. There was always something that surprised you.",
"One manager concluded: You get no discount. You can never go the shorter way. There was always something that surprised you. We thought this was a lot like a three headed monster, so when you chopped off one of its heads, three other emerged, every solution was followed by more problems. The health professionals who volunteered to join ETT did so for different reasons. Ebola preparedness was 'a job that had to be done', and 'someone had to do it'.",
"The health professionals who volunteered to join ETT did so for different reasons. Ebola preparedness was 'a job that had to be done', and 'someone had to do it'. Some referred to ethical or professional obligations: This is just a part of being a nurse, to encounter situations that can be dangerous to you or someone else, but you have made this decision and you deal with it. Some connected their decision to their 'action gene' or 'addiction to taking risks', while others said they had already raised their kids and had years of experience, including work with other epidemics, such as HIV. Yet, the practice of volunteering in the preparation was questioned. One participant said: We learned that we could not rely on volunteers … when you work in an infectious disease department you cannot choose what infections you want to work with.",
"Yet, the practice of volunteering in the preparation was questioned. One participant said: We learned that we could not rely on volunteers … when you work in an infectious disease department you cannot choose what infections you want to work with. ER staff indicated that for them working in the ER was enough of a risk to take, no reason to expose oneself even more by joining the ETT, and appreciated that others had volunteered. All participants noted that co-operation and communication had generally functioned well during the preparedness planning, with information flowing both ways. Short communication lines within the healthcare system were perceived as both a strength and a weakness; a strength, insofar as people knew each other, but a weakness because of the uneven burden of workload. Staff of the ETT and in the ER felt they had been well-informed, and that openness and honesty had characterized the planning and diminished their initial fear.",
"Short communication lines within the healthcare system were perceived as both a strength and a weakness; a strength, insofar as people knew each other, but a weakness because of the uneven burden of workload. Staff of the ETT and in the ER felt they had been well-informed, and that openness and honesty had characterized the planning and diminished their initial fear. Those in managerial positions had listened and taken their opinions into consideration. One said: They were honest, no one was hiding anything, everything was on the table, no one tried to make things more appealing and say that everything would be OK, they just told us about things as they were. Both management and participants from the ETT and ER expressed their ambiguity in terms of trust, doubt, and fear. Participants conveyed trust in the health system and their own role as health professionals, while at the same time admitting to facing formidable challenges during the elaboration of the preparedness plan.",
"Both management and participants from the ETT and ER expressed their ambiguity in terms of trust, doubt, and fear. Participants conveyed trust in the health system and their own role as health professionals, while at the same time admitting to facing formidable challenges during the elaboration of the preparedness plan. Facilities for isolation and treatment of patients with Ebola were less than perfect: We assessed how we could use the department … and change it in just a few hours into some kind of an isolation unit that we could possibly use. Some compared this short-term isolation facility to a 'camping site', as the facilities were too provisional and not comparable to those found elsewhere. There was also doubt about how many Ebola patients LSH would be able to care for: 'Maybe one or two patients, barely more'. Respondents believed that the training and education of the members of the ETT and ER had been satisfactory.",
"There was also doubt about how many Ebola patients LSH would be able to care for: 'Maybe one or two patients, barely more'. Respondents believed that the training and education of the members of the ETT and ER had been satisfactory. They felt that it had been proportionate to the risk, while some were concerned about the lack of staff. Nonetheless, there were contradictions on the division of labour among the professionals, exemplified by different ideas on how to proceed if a patient suspected of having an EVD came in an ambulance to the LSH for treatment. Almost all participants stated that they were ready to do their part in the Ebola response, or 'as ready as we could be'. There were diverse opinions on what it meant to be ready: to treat one confirmed case of Ebola, one suspected case, or more EVD patients?",
"Almost all participants stated that they were ready to do their part in the Ebola response, or 'as ready as we could be'. There were diverse opinions on what it meant to be ready: to treat one confirmed case of Ebola, one suspected case, or more EVD patients? When asked if Ebola was a real threat to the country, participants usually referred to how easy it was to travel the globe: 'Yeah, why not, the world is getting smaller'. Although Ebola was thought of as a real danger by many, some participants expressed difficulty in taking their training seriously, doubting that Ebola would ever reach Iceland. One respondent said: People were dedicated in the beginning, but when the news appeared that Ebola was receding, that diminished, and I never felt like this formally ended. Participants described their relief that nothing really happened, while emphasizing the need to experience a real situation to evaluate the preparedness efforts.",
"One respondent said: People were dedicated in the beginning, but when the news appeared that Ebola was receding, that diminished, and I never felt like this formally ended. Participants described their relief that nothing really happened, while emphasizing the need to experience a real situation to evaluate the preparedness efforts. One participant said that 'a little bit more seriousness would have been needed in the PPE practices'. It was taken as a manifestation of fear that some of the staff in the communicable disease department of the LSH refused to take part in the ETT. When describing their fears, ETT members frequently connected it to their working conditions. Many of them were afraid that they would not get the best PPE, others that they would not do the donning/doffing correctly and, lastly, they were worried about work performance while in the PPE.",
"When describing their fears, ETT members frequently connected it to their working conditions. Many of them were afraid that they would not get the best PPE, others that they would not do the donning/doffing correctly and, lastly, they were worried about work performance while in the PPE. One participant said: What bothered most of us was how uncomfortable the PPE was and I think that made people nervous: \"How will I manage working in this for hours?\" Another described the donning/doffing process like a 'complicated ballroom dance'. Moreover, participants were afraid of 'unknown territories', that is, they did not know the hospital ward, they were supposed to work in, and some team members had no recent experience of clinical work. One participant said: I didn't think these non-clinical people belonged in the team, because this is a very clinical environment in addition to having to be in this costume PPE with the risk of becoming infected by mistake.",
"Moreover, participants were afraid of 'unknown territories', that is, they did not know the hospital ward, they were supposed to work in, and some team members had no recent experience of clinical work. One participant said: I didn't think these non-clinical people belonged in the team, because this is a very clinical environment in addition to having to be in this costume PPE with the risk of becoming infected by mistake. Those with non-clinical background were, however, aware of their limitations: I realized that I would not be the one in the front, I would not be managing patients directly. The importance ascribed to teamwork was evident in relation to fear. Participants described fear of working with people they had not worked with before: The weakest link in the preparation was that even though I knew their faces, I had never worked with them. Another issue was no-show by some team members in training sessions or in lectures: This is team-work, one does this and the other one does this, we help each other.",
"Participants described fear of working with people they had not worked with before: The weakest link in the preparation was that even though I knew their faces, I had never worked with them. Another issue was no-show by some team members in training sessions or in lectures: This is team-work, one does this and the other one does this, we help each other. Then you don't want to be working with someone who didn't show up. There were a lot of doctors who just dropped in, dropped out, and then dropped in again. I asked myself: Are these individuals … ready to take this on? Participants in the ETT mentioned the precautions they took or intended to take to cope with their feelings of fear, should Ebola emerge in Iceland.",
"I asked myself: Are these individuals … ready to take this on? Participants in the ETT mentioned the precautions they took or intended to take to cope with their feelings of fear, should Ebola emerge in Iceland. A major precaution was planning to avoid contact with the family while working with Ebola patients. One participant said: 'You thought … about your children at school … parents in the neighbourhood …' if they knew s he was working with an Ebola patient. For them, it was important they would have access to special accommodation in case of clinical EVD work 'so I wouldn't be exposing anyone or creating hysteria'. ETT members mentioned the extra insurance offered as a prerequisite for taking part in the team.",
"For them, it was important they would have access to special accommodation in case of clinical EVD work 'so I wouldn't be exposing anyone or creating hysteria'. ETT members mentioned the extra insurance offered as a prerequisite for taking part in the team. 'The normal insurance for LHS staff would not cover everything if we were to become sick or even lose our lives.' Amongst ER staff, the matter of insurance did seem to be less of an issue compared to the ETT. One respondent said: 'You are used to being at risk by many disease threats'. Furthermore, the issue of higher salaries and risk commission came up in the interviews, but overall did not matter as much to the participants as the insurance, or assurance of accommodation in case of need.",
"One respondent said: 'You are used to being at risk by many disease threats'. Furthermore, the issue of higher salaries and risk commission came up in the interviews, but overall did not matter as much to the participants as the insurance, or assurance of accommodation in case of need. Characteristics associated with Iceland and the Icelandic people were referred to repeatedly by participants. The concept 'Tiny Iceland' was often mentioned and emerged with positive and negative connotations. 'Tiny Iceland' referred to the size of the country and population and its perceived capability to still 'get the job done'. even though compromises had to be made.",
"'Tiny Iceland' referred to the size of the country and population and its perceived capability to still 'get the job done'. even though compromises had to be made. Comparing how Iceland handled its responsibilities differently from other countries of a larger size was often brought up, both with pride in Iceland as a strong independent nation, and with insecurities about its capacity in comparison to other countries. It was pointed out that since the preparedness process was in the hands of a few people, everyone knew their role. As one administrator said: This little hospital system, as complicated as it might seem every day, gives you the chance to just pick up the phone and call the one in charge. Being a small population presents challenges regarding resources, infrastructure, and specialized medical training to comply with standards of international actors.",
"As one administrator said: This little hospital system, as complicated as it might seem every day, gives you the chance to just pick up the phone and call the one in charge. Being a small population presents challenges regarding resources, infrastructure, and specialized medical training to comply with standards of international actors. Notions of Icelanders as resilient in spite of shortcomings were common; referring to the experience of preparedness planning and training, one health staff said: It was very much the Icelandic way, we'll manage, we'll work it out, and there was so much ingenuity. This notion of a particular Icelandic approach to coping, in spite of shortcomings, was also detected more generally, as in the statement: Would it have worked? Yes, it would have worked. Would it have been optimal?",
"Yes, it would have worked. Would it have been optimal? We cannot say, it would have been optimal; we can say, it would have been sufficient. In contrast to this, there were concerns about whether Icelandic aid workers falling ill in Ebolaaffected countries should be transferred to Iceland or to hospitals in other Nordic countries with better isolation units. Some of the participants trusted that patients with EVD would not be transferred to Iceland. One participant stated: You heard that Norwegians were criticized for transferring their aid worker from Africa to Norway. We don't know what would have happened if they would have transferred an Icelander into the country.",
"One participant stated: You heard that Norwegians were criticized for transferring their aid worker from Africa to Norway. We don't know what would have happened if they would have transferred an Icelander into the country. We don't have good enough isolation unitsyou are not supposed to send patients to a hospital that is less than 100%. I thought there was assurance in that. During the devastating Ebola epidemic in West Africa that spread to neighbouring sub-Saharan countries, North America, and Europe , preparedness plans were widely elaborated and later evaluated. Evaluations have, for example, been conducted in 11 African countries close to the epidemic , in the EU region , and the US .",
"During the devastating Ebola epidemic in West Africa that spread to neighbouring sub-Saharan countries, North America, and Europe , preparedness plans were widely elaborated and later evaluated. Evaluations have, for example, been conducted in 11 African countries close to the epidemic , in the EU region , and the US . Here we present data from a qualitative case study on the process, and experiences with establishing a preparedness plan for Ebola in Iceland in 2014. Interviews with staff who were engaged, either as administrators or frontline healthcare workers, alert us to the manner in which geographic, demographic, cultural, and organizational characteristics shaped the response. The results show that the process of establishing and training for preparedness was permeated by ambiguities of pride and pragmatism, trust, doubts, and fear. 'Getting the job done' theme 1 refers to the multitude of tasks and considerations that surrounds and feeds into the preparedness plan itself and are necessary for successful planning and implementation.",
"The results show that the process of establishing and training for preparedness was permeated by ambiguities of pride and pragmatism, trust, doubts, and fear. 'Getting the job done' theme 1 refers to the multitude of tasks and considerations that surrounds and feeds into the preparedness plan itself and are necessary for successful planning and implementation. Using the metaphors of 'hard core' and 'soft periphery', Langley and Denis emphasize the importance of relatively 'peripheral' concerns and processes for planning and implementation of new interventions. The hard core represents the actual intervention or goal, e.g. implementation of a preparedness plan. The soft periphery refers to all the contextually important networking, negotiations, and agreements necessary to deliver the hard core.",
"implementation of a preparedness plan. The soft periphery refers to all the contextually important networking, negotiations, and agreements necessary to deliver the hard core. If the soft periphery is neglected, it will cause multiple challenges in the implementation process, and the benefit of the hard core, the intervention itself, may not transpire as anticipated. Due attention to the soft periphery may, however, considerably promote the delivery of an innovation, and secure support from important stakeholders. In our data, one manager speaks of the preparedness process as dealing with a three-headed monster where every solution was followed by new problems. The data indicate that the process of dealing with 'the three headed monster' was given due attention as a means to successfully develop Iceland's preparedness plan.",
"In our data, one manager speaks of the preparedness process as dealing with a three-headed monster where every solution was followed by new problems. The data indicate that the process of dealing with 'the three headed monster' was given due attention as a means to successfully develop Iceland's preparedness plan. Comprehensive consultations and the involvement of many associated institutions were mentioned. Still ambiguity remained with some staff in terms of division of responsibilities and taskse.g. when transporting a patient potentially infected with Ebola from the airport to the hospital, and other such activities. During epidemics, rumours, gossip, and unreliable information on the news and social media spread rapidly, resulting in so-called 'infodemics' .",
"when transporting a patient potentially infected with Ebola from the airport to the hospital, and other such activities. During epidemics, rumours, gossip, and unreliable information on the news and social media spread rapidly, resulting in so-called 'infodemics' . The West African Ebola epidemic was covered widely by media , and the fear of Ebola reached every corner of the world, exemplified by travel bans from affected countries, and trade barriers , in contrast to the ongoing epidemic in the Democratic Republic of Congo . In our second theme, trust, doubt, and fear of health workers were represented. Although all intentions were good, concerns remained about the suitability and safety of the isolation ward, the PPE, and other tools, as well as adequate engagement of colleagues who might potentially work alongside them, in case an Ebola patient came to Iceland. The foreignness of putting on, removing, and working from within a PPE and the trustworthiness of available PPE were mentioned.",
"Although all intentions were good, concerns remained about the suitability and safety of the isolation ward, the PPE, and other tools, as well as adequate engagement of colleagues who might potentially work alongside them, in case an Ebola patient came to Iceland. The foreignness of putting on, removing, and working from within a PPE and the trustworthiness of available PPE were mentioned. In preparedness efforts in other countries, scarcity of resources in relation to manpower demand and problems with training and protocols involving PPE were common challenges . Similar problems were encountered in Iceland. Provisory treatment facility had to be designed, called 'camping site' by some, in contrast to facilities found elsewhere . Further, the ETT was established based on voluntary recruitment rather than on the staff's assigned roles within the healthcare system, a procedure that was deemed less than optimal.",
"Provisory treatment facility had to be designed, called 'camping site' by some, in contrast to facilities found elsewhere . Further, the ETT was established based on voluntary recruitment rather than on the staff's assigned roles within the healthcare system, a procedure that was deemed less than optimal. The members of the ETT pointed out that they had never worked together as a team under circumstances that demanded strict adherence to infectious control procedures. This eroded trust, compounded by the laissez-faire attitude of some of its members during the preparation exercises, possibly due to other competing tasks in a busy hospital and insufficient resources that hampered full participation . Further, it was a constraint that simulation exercises were not an option, found to be an important element in preparation for epidemics . This might have resulted in less than optimal staff protection for those who would have been in direct contact with an infected patient, as reported during the SARS epidemic in Canada .",
"Further, it was a constraint that simulation exercises were not an option, found to be an important element in preparation for epidemics . This might have resulted in less than optimal staff protection for those who would have been in direct contact with an infected patient, as reported during the SARS epidemic in Canada . Anthropological work on emergency preparedness emphasizes the connectedness between health professionals, technological devices, and knowledge as a prerequisite for successful preparedness. Wolf and Hall present preparedness efforts as a form of governance that involves human bodies those of health professionals , clinical architectures e.g. isolation wards , and technical artefacts gloves, protective suits, disinfectants, etc. .",
"isolation wards , and technical artefacts gloves, protective suits, disinfectants, etc. . During preparedness training and implementation, 'nursing bodies are transformed into instruments of preparedness', and become part of infrastructural arrangements. Health professionals are, here, both vulnerable and powerful tools in the management of contamination. The authors argue that successful planning, training, and implementation of a preparedness plan require such intrinsic connectedness. In the case of Ebola preparedness in Iceland, health professionals draw our attention to dilemmas of connectedness, and their assessment of the fact that these shortcomings might hamper the mobilization of 'preparedness within the human body'that is, the embodied experience, routine, and tacit knowledge which Wolf and Hall state are key to successful implementation.",
"The authors argue that successful planning, training, and implementation of a preparedness plan require such intrinsic connectedness. In the case of Ebola preparedness in Iceland, health professionals draw our attention to dilemmas of connectedness, and their assessment of the fact that these shortcomings might hamper the mobilization of 'preparedness within the human body'that is, the embodied experience, routine, and tacit knowledge which Wolf and Hall state are key to successful implementation. Repeated enactment of receiving and treating a patient with Ebola within experienced and trustful teams would probably enhance such embodiment, provided that there is justified trust in the involved technology. In addition, repetition would also strengthen the 'soft periphery' of preparedness, and divisions of responsibilities would be clearer manifested. In the third theme, we observe how notions of the 'Icelandic way' help participants make sense of ambiguities about Ebola preparedness. Loftsdóttir explored how people negotiated the imagination of the local and the global during the 2008 economic crisis in Iceland .",
"In the third theme, we observe how notions of the 'Icelandic way' help participants make sense of ambiguities about Ebola preparedness. Loftsdóttir explored how people negotiated the imagination of the local and the global during the 2008 economic crisis in Iceland . Notions of the intrinsic character of Iceland, and of being Icelandic, serve to underscore certain points and explain positive and negative experiences with the preparedness plan. Iceland is far away from the continents, but still connected through global needs for policy, risk of contamination, and dependency in terms of collaboration, in emergencies emerging from elsewhere. In our study, participants highlighted the importance of believing in oneself and the 'Icelandic way of doing things,' summed up in the paraphrase 'þetta reddast' things always have a way of working out in the end . The preparedness plan had to be completed, and adapted to Iceland's particular global situation.",
"In our study, participants highlighted the importance of believing in oneself and the 'Icelandic way of doing things,' summed up in the paraphrase 'þetta reddast' things always have a way of working out in the end . The preparedness plan had to be completed, and adapted to Iceland's particular global situation. In the 21st century, the world has faced new epidemic threats, such as SARS, and old scourges such as the plague have resurfaced . One of the main findings on Ebola preparedness measures in the EU was that measures taken were based on past preparedness and experience of other epidemics, such as SARS and H1N1 . Further, key stakeholders within each country found their measures to have been adequate for dealing with a single case of Ebola, as was the case in Iceland. A preparedness plan for pandemic influenzae in Iceland was elaborated in 2006activated in response to the H1N1 epidemic in 2009and revised in 2016 .",
"Further, key stakeholders within each country found their measures to have been adequate for dealing with a single case of Ebola, as was the case in Iceland. A preparedness plan for pandemic influenzae in Iceland was elaborated in 2006activated in response to the H1N1 epidemic in 2009and revised in 2016 . During the elaboration of these plans, communication among the different levels of the healthcare system and supporting agencies, such as the DCPEM, had been clearly defined, and proved to be useful in the preparedness for Ebola. Further, as found important in preparedness activities for pandemic influenzae elsewhere , honesty, transparency in communication, and sharing of information from managers to front-line health professionals, was found to be critical. It gave a feeling of being involved, and mitigated the fear that is so frequently encountered during epidemics . Iceland was far away from the epicentre of the Ebola epidemic in West Africa.",
"It gave a feeling of being involved, and mitigated the fear that is so frequently encountered during epidemics . Iceland was far away from the epicentre of the Ebola epidemic in West Africa. Yet this case study shows that health professionals felt the strain of possibly having to treat one or more patients with EVD. Their situation stands in sharp contrast to the situation in the three worst affected West African countries that lacked staff, stuff, space, and systems to effectively address the challenge of EVD. Although Icelandic health professionals had trust in the national healthcare system, and in their own capacity, doubt and fear influenced the reflections on preparedness planning of both administrators and healthcare staff. References to national identity and the characteristic of an 'Icelandic approach' to handling challenges assisted participants in coming to terms with the experienced shortcomings of the preparedness plan, and underscored the pride in the ingenuity applied in the process.",
"Although Icelandic health professionals had trust in the national healthcare system, and in their own capacity, doubt and fear influenced the reflections on preparedness planning of both administrators and healthcare staff. References to national identity and the characteristic of an 'Icelandic approach' to handling challenges assisted participants in coming to terms with the experienced shortcomings of the preparedness plan, and underscored the pride in the ingenuity applied in the process. These references negotiate the role and character of the nation of Iceland, and its role in a globalized world, as both a small and isolated nation on one hand, and a central and capable one, on the other. The experienced ambiguity needs attention in a health system and among healthcare staff that have to act resolutely and unfailingly, should they be placed in charge of containing contamination. This study points to the necessity of repeatedly re-enacting, as realistically as possible, the likely scenarios of receiving and treating one or more patients infected with Ebola or other contagious global health threats as a routine matter. This would assist in the identification of overlooked 'soft periphery' concerns, and promote embodied preparedness among teams of health care staff on the frontline.",
"This study points to the necessity of repeatedly re-enacting, as realistically as possible, the likely scenarios of receiving and treating one or more patients infected with Ebola or other contagious global health threats as a routine matter. This would assist in the identification of overlooked 'soft periphery' concerns, and promote embodied preparedness among teams of health care staff on the frontline. Geir Gunnlaugsson conceptualized the study, and took part in all necessary steps towards its completion, such as analysis and interpretation of data, and writing the manuscript for submission. Íris Eva Hauksdóttir collected and analysed the data as part of a master thesis work conducted under the supervision of all three co-authors, revised the manuscript, and approved the final version. Ib Bygbjerg took part in the interpretation of data, revision of the manuscript, and approved the final version. Britt Pinkowski Tersbøl took part in designing interview tools and in the thematic analysis of interview data, interpretation, revision of the manuscript, and approved the final version.",
"Ib Bygbjerg took part in the interpretation of data, revision of the manuscript, and approved the final version. Britt Pinkowski Tersbøl took part in designing interview tools and in the thematic analysis of interview data, interpretation, revision of the manuscript, and approved the final version. Dr. Gunnlaugsson reports he was the Chief Medical Officer CMO for Iceland, Directorate of Health, in the period 2010-2014. Other authors report no conflict of interest. The study was reported to the Data Protection Authority and approved by the National Bioethics Committee in Iceland number VSI- . Subsequently, the study was approved by the University Hospital Ethical Committee on 4 February 2016 number LSH .",
"The study was reported to the Data Protection Authority and approved by the National Bioethics Committee in Iceland number VSI- . Subsequently, the study was approved by the University Hospital Ethical Committee on 4 February 2016 number LSH . Participants signed an informed consent form before taking part in the study. Not applicable. The manuscript builds on the work of Íris Eva Hauksdóttir towards a MSc in Global Health, Section of Global Health, Department of Public Health, Copenhagen University, Denmark."
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What are the prerequisites for successful emergency preparedness for an epidemic? | connectedness between health professionals, technological devices, and knowledge | [
"Background: The Ebola epidemic in West Africa caused global fear and stirred up worldwide preparedness activities in countries sharing borders with those affected, and in geographically far-away countries such as Iceland. Objective: To describe and analyse Ebola preparedness activities within the Icelandic healthcare system, and to explore the perspectives and experiences of managers and frontline health workers. Methods: A qualitative case study, based on semi-structured interviews with 21 staff members in the national Ebola Treatment Team, Emergency Room at Landspitali University Hospital, and managers of the response team. Results: Contextual factors such as culture and demography influenced preparedness, and contributed to the positive state of mind of participants, and ingenuity in using available resources for preparedness. While participants believed they were ready to take on the task of Ebola, they also had doubts about the chances of Ebola ever reaching Iceland. Yet, factors such as fear of Ebola and the perceived stigma associated with caring for a potentially infected Ebola patient, influenced the preparation process and resulted in plans for specific precautions by staff to secure the safety of their families.",
"While participants believed they were ready to take on the task of Ebola, they also had doubts about the chances of Ebola ever reaching Iceland. Yet, factors such as fear of Ebola and the perceived stigma associated with caring for a potentially infected Ebola patient, influenced the preparation process and resulted in plans for specific precautions by staff to secure the safety of their families. There were also concerns about the teamwork and lack of commitment by some during training. Being a ‘tiny’ nation was seen as both an asset and a weakness in the preparation process. Honest information sharing and scenario-based training contributed to increased confidence amongst participants in the response plans. Conclusions: Communication and training were important for preparedness of health staff in Iceland, in order to receive, admit, and treat a patient suspected of having Ebola, while doubts prevailed on staff capacity to properly do so.",
"Honest information sharing and scenario-based training contributed to increased confidence amongst participants in the response plans. Conclusions: Communication and training were important for preparedness of health staff in Iceland, in order to receive, admit, and treat a patient suspected of having Ebola, while doubts prevailed on staff capacity to properly do so. For optimal preparedness, likely scenarios for future global security health threats need to be repeatedly enacted, and areas plagued by poverty and fragile healthcare systems require global support. Text: Global health; prevention and control; public policy; qualitative evaluation; emergency responders; communicable diseases; emerging; fear Background On 8 August 2014, the World Health Organization declared the Ebola epidemic in West Africa as a Public Health Emergency of International Concern PHEIC under the International Health Regulations IHR . All three of the worst affected countries were to address the emerging epidemic challenge without staff, stuff, space and systems . With the epidemic seemingly out of control, and a proportionately high number of doctors, nurses, and midwives succumbing to Ebola , there was a growing fear of transmission beyond the region.",
"All three of the worst affected countries were to address the emerging epidemic challenge without staff, stuff, space and systems . With the epidemic seemingly out of control, and a proportionately high number of doctors, nurses, and midwives succumbing to Ebola , there was a growing fear of transmission beyond the region. In breach of WHO recommendations and guidelines , flights were cancelled and cross-border movement curtailed . The epidemic caused public concern outside West Africa , as fear and racism found fertile ground , and in an effort to stop the international spread of the disease, all states were advised to be prepared to detect, investigate, and manage Ebola cases . Preparedness as part of disaster risk reduction is defined as 'the knowledge and capacities developed by governments, response and recovery organizations, communities and individuals to effectively anticipate, respond to, and recover from the impacts of likely, imminent or current disasters' . Yet, preparedness is also enveloped in and influenced by the socio-cultural dimension at the individual, organizational, and national levels, and measures to manage outbreaks are not always accepted or accommodated by the communities to which they are applied .",
"Preparedness as part of disaster risk reduction is defined as 'the knowledge and capacities developed by governments, response and recovery organizations, communities and individuals to effectively anticipate, respond to, and recover from the impacts of likely, imminent or current disasters' . Yet, preparedness is also enveloped in and influenced by the socio-cultural dimension at the individual, organizational, and national levels, and measures to manage outbreaks are not always accepted or accommodated by the communities to which they are applied . An analysis of eight European countries' preparedness plans since 2009 for countering a future influenza A H1N1 pandemic revealed that the way plans were framed varied considerably, and ' told us something about how the different countries want pandemics and preparedness to be understood by the public' . More research was encouraged into cultural and social structures in the respective countries. In Iceland, information about the Ebola epidemic in West Africa came from several sources. The Directorate of Health DH first reported on the epidemic on 8 April 2014 .",
"In Iceland, information about the Ebola epidemic in West Africa came from several sources. The Directorate of Health DH first reported on the epidemic on 8 April 2014 . In Icelandic media, the rapid progress of the Ebola epidemic in West Africa was increasingly highlighted, and exported Ebola cases to Spain, USA, and elsewhere, were widely covered. Fear of a global epidemic was rife, and in media and online discussions, doubts were raised about the Icelandic health system´s capacity to take care of a patient with Ebola , despite its ranking as one of the best in the world . On 11 August 2014, three days after WHO declared PHEIC because of Ebola, DH encouraged Icelandic citizens to avoid visits to the area, if possible, and reported that the national epidemic preparedness plan was being activated for Ebola . It was elaborated by a team that involved the Chief Epidemiologist at the DH, Landspitali University Hospital LSH , the Department of Civil Protection and Emergency Management DCPEM , and the seven Primary Healthcare Regional Organizations in the country at the time.",
"On 11 August 2014, three days after WHO declared PHEIC because of Ebola, DH encouraged Icelandic citizens to avoid visits to the area, if possible, and reported that the national epidemic preparedness plan was being activated for Ebola . It was elaborated by a team that involved the Chief Epidemiologist at the DH, Landspitali University Hospital LSH , the Department of Civil Protection and Emergency Management DCPEM , and the seven Primary Healthcare Regional Organizations in the country at the time. Key external partners were the European Centre for Disease Prevention and Control ECDC and WHO, in addition to Nordic collaborators in epidemic preparedness . At the same time, it was regarded as highly unlikely that Ebola Virus Disease EVD would spread in the country . Recognized scenarios included the possible appearance of an infected person in need of treatment, who could be either an Icelandic citizen who had visited or worked in one of the affected West African countries, or a person with signs of EVD on a trans-Atlantic flight in the navigation area controlled by Icelandic authorities . On 3 November 2014, the plan was put to the test when a foreign airline made a non-scheduled landing at Keflavík International Airport due to fear of EVD in one passenger from South Africa.",
"Recognized scenarios included the possible appearance of an infected person in need of treatment, who could be either an Icelandic citizen who had visited or worked in one of the affected West African countries, or a person with signs of EVD on a trans-Atlantic flight in the navigation area controlled by Icelandic authorities . On 3 November 2014, the plan was put to the test when a foreign airline made a non-scheduled landing at Keflavík International Airport due to fear of EVD in one passenger from South Africa. Parked in a closed-off area, a physician in full Personal Protective Equipment PPE entered the plane, but quickly ruled out Ebola . Irrespective of good or bad overall performance, health systems are tested in times of crisis, such as epidemics. Here, the aim is to describe and analyse the process of establishing preparedness plans for Ebola in Iceland, with a specific focus on the perspectives and experiences of managers and frontline health workers involved in the process. This study is part of a larger study on the impact that the global threat of the Ebola epidemic had in Iceland .",
"Here, the aim is to describe and analyse the process of establishing preparedness plans for Ebola in Iceland, with a specific focus on the perspectives and experiences of managers and frontline health workers involved in the process. This study is part of a larger study on the impact that the global threat of the Ebola epidemic had in Iceland . Qualitative case study methodology was applied, perceiving the preparedness planning and training process as the case with clear boundaries of the initiation, process, and wrap-up of preparedness planning and training. The study was conducted in April-May 2016, and the interviewed participants were administrators and frontline health professionals central to the case, so as to explore their perspectives and experiences concerning Ebola preparedness . Staff in managerial positions were contacted by one of the authors GG for permission to interview them based on their role in the preparedness plan. To identify potential interviewees in the Ebola Treatment Team ETT , the director of the team listed relevant email contacts.",
"Staff in managerial positions were contacted by one of the authors GG for permission to interview them based on their role in the preparedness plan. To identify potential interviewees in the Ebola Treatment Team ETT , the director of the team listed relevant email contacts. Those who responded positively were subsequently invited for an interview, conducted in Icelandic by one of the authors ÍEH , a physiotherapist. In case interviewees suggested other potential participants, they were invited through email to participate. A similar methodology was applied to identify participants from the Emergency Room ER . They were included in order to represent frontline health workers who worked in the only ER in Reykjavík, where persons exposed to EVD were most likely to first seek care in case of acute illness.",
"A similar methodology was applied to identify participants from the Emergency Room ER . They were included in order to represent frontline health workers who worked in the only ER in Reykjavík, where persons exposed to EVD were most likely to first seek care in case of acute illness. Three separate interview guides were developedone each for managers, ETT, and ER respectively see supplementary material . The interviews included open questions probing the role of their institution in preparedness, the experience of the training process, challenges encountered or expected, and any dilemmas that they may have experienced in relation to the preparedness plan. The recruitment of participants was concluded when saturation was reached. Each interview was recorded and took about 20 to 60 minutes; they were then transcribed and analysed using thematic analysis.",
"The recruitment of participants was concluded when saturation was reached. Each interview was recorded and took about 20 to 60 minutes; they were then transcribed and analysed using thematic analysis. The data material was read through repeatedly, sorted, and categorized, based on the participants' priorities in the representation of their views. From this exercise, three broad themes were inductively identified that corresponded to critical perspectives introduced by the participants. Permission to conduct the study was granted by Iceland's National Bioethics Committee VSN- and Landspitali University Hospital LSH 13-16, 4 February 2016 . Reporting on the results was guided by the COREC guidelines ; however, to ensure anonymity of the respondents within the small community of staff who took part in the preparedness activities, participant information is not associated to quotations.",
"Permission to conduct the study was granted by Iceland's National Bioethics Committee VSN- and Landspitali University Hospital LSH 13-16, 4 February 2016 . Reporting on the results was guided by the COREC guidelines ; however, to ensure anonymity of the respondents within the small community of staff who took part in the preparedness activities, participant information is not associated to quotations. The Icelandic Ebola Preparedness Plan included the establishment of an ETT within LSH , and the preparatory activities engaged more than two hundred staff across all of its departments. The ETT consisted of about 50 healthcare professionals who had volunteered to participate, including 11 doctors and 28 nurses, a few laboratory technicians, radiologists, and auxiliary nurses. They attended special training sessions focused on protocols for admission and treatment of a patient with EVD, the donning/doffing of PPE, and personal protective measures during patient care. A new provisory unit was designed to be set up on the ground floor to minimize the risk of infection spreading to other units within the hospital, with two rooms specifically identified for the care of a patient with EVD .",
"They attended special training sessions focused on protocols for admission and treatment of a patient with EVD, the donning/doffing of PPE, and personal protective measures during patient care. A new provisory unit was designed to be set up on the ground floor to minimize the risk of infection spreading to other units within the hospital, with two rooms specifically identified for the care of a patient with EVD . Managers' accounts of this period elaborated the complexity of preparedness planning in terms of the involved institutions, actors, procedures and requirement of the plan. One manager concluded: You get no discount. You can never go the shorter way. There was always something that surprised you.",
"One manager concluded: You get no discount. You can never go the shorter way. There was always something that surprised you. We thought this was a lot like a three headed monster, so when you chopped off one of its heads, three other emerged, every solution was followed by more problems. The health professionals who volunteered to join ETT did so for different reasons. Ebola preparedness was 'a job that had to be done', and 'someone had to do it'.",
"The health professionals who volunteered to join ETT did so for different reasons. Ebola preparedness was 'a job that had to be done', and 'someone had to do it'. Some referred to ethical or professional obligations: This is just a part of being a nurse, to encounter situations that can be dangerous to you or someone else, but you have made this decision and you deal with it. Some connected their decision to their 'action gene' or 'addiction to taking risks', while others said they had already raised their kids and had years of experience, including work with other epidemics, such as HIV. Yet, the practice of volunteering in the preparation was questioned. One participant said: We learned that we could not rely on volunteers … when you work in an infectious disease department you cannot choose what infections you want to work with.",
"Yet, the practice of volunteering in the preparation was questioned. One participant said: We learned that we could not rely on volunteers … when you work in an infectious disease department you cannot choose what infections you want to work with. ER staff indicated that for them working in the ER was enough of a risk to take, no reason to expose oneself even more by joining the ETT, and appreciated that others had volunteered. All participants noted that co-operation and communication had generally functioned well during the preparedness planning, with information flowing both ways. Short communication lines within the healthcare system were perceived as both a strength and a weakness; a strength, insofar as people knew each other, but a weakness because of the uneven burden of workload. Staff of the ETT and in the ER felt they had been well-informed, and that openness and honesty had characterized the planning and diminished their initial fear.",
"Short communication lines within the healthcare system were perceived as both a strength and a weakness; a strength, insofar as people knew each other, but a weakness because of the uneven burden of workload. Staff of the ETT and in the ER felt they had been well-informed, and that openness and honesty had characterized the planning and diminished their initial fear. Those in managerial positions had listened and taken their opinions into consideration. One said: They were honest, no one was hiding anything, everything was on the table, no one tried to make things more appealing and say that everything would be OK, they just told us about things as they were. Both management and participants from the ETT and ER expressed their ambiguity in terms of trust, doubt, and fear. Participants conveyed trust in the health system and their own role as health professionals, while at the same time admitting to facing formidable challenges during the elaboration of the preparedness plan.",
"Both management and participants from the ETT and ER expressed their ambiguity in terms of trust, doubt, and fear. Participants conveyed trust in the health system and their own role as health professionals, while at the same time admitting to facing formidable challenges during the elaboration of the preparedness plan. Facilities for isolation and treatment of patients with Ebola were less than perfect: We assessed how we could use the department … and change it in just a few hours into some kind of an isolation unit that we could possibly use. Some compared this short-term isolation facility to a 'camping site', as the facilities were too provisional and not comparable to those found elsewhere. There was also doubt about how many Ebola patients LSH would be able to care for: 'Maybe one or two patients, barely more'. Respondents believed that the training and education of the members of the ETT and ER had been satisfactory.",
"There was also doubt about how many Ebola patients LSH would be able to care for: 'Maybe one or two patients, barely more'. Respondents believed that the training and education of the members of the ETT and ER had been satisfactory. They felt that it had been proportionate to the risk, while some were concerned about the lack of staff. Nonetheless, there were contradictions on the division of labour among the professionals, exemplified by different ideas on how to proceed if a patient suspected of having an EVD came in an ambulance to the LSH for treatment. Almost all participants stated that they were ready to do their part in the Ebola response, or 'as ready as we could be'. There were diverse opinions on what it meant to be ready: to treat one confirmed case of Ebola, one suspected case, or more EVD patients?",
"Almost all participants stated that they were ready to do their part in the Ebola response, or 'as ready as we could be'. There were diverse opinions on what it meant to be ready: to treat one confirmed case of Ebola, one suspected case, or more EVD patients? When asked if Ebola was a real threat to the country, participants usually referred to how easy it was to travel the globe: 'Yeah, why not, the world is getting smaller'. Although Ebola was thought of as a real danger by many, some participants expressed difficulty in taking their training seriously, doubting that Ebola would ever reach Iceland. One respondent said: People were dedicated in the beginning, but when the news appeared that Ebola was receding, that diminished, and I never felt like this formally ended. Participants described their relief that nothing really happened, while emphasizing the need to experience a real situation to evaluate the preparedness efforts.",
"One respondent said: People were dedicated in the beginning, but when the news appeared that Ebola was receding, that diminished, and I never felt like this formally ended. Participants described their relief that nothing really happened, while emphasizing the need to experience a real situation to evaluate the preparedness efforts. One participant said that 'a little bit more seriousness would have been needed in the PPE practices'. It was taken as a manifestation of fear that some of the staff in the communicable disease department of the LSH refused to take part in the ETT. When describing their fears, ETT members frequently connected it to their working conditions. Many of them were afraid that they would not get the best PPE, others that they would not do the donning/doffing correctly and, lastly, they were worried about work performance while in the PPE.",
"When describing their fears, ETT members frequently connected it to their working conditions. Many of them were afraid that they would not get the best PPE, others that they would not do the donning/doffing correctly and, lastly, they were worried about work performance while in the PPE. One participant said: What bothered most of us was how uncomfortable the PPE was and I think that made people nervous: \"How will I manage working in this for hours?\" Another described the donning/doffing process like a 'complicated ballroom dance'. Moreover, participants were afraid of 'unknown territories', that is, they did not know the hospital ward, they were supposed to work in, and some team members had no recent experience of clinical work. One participant said: I didn't think these non-clinical people belonged in the team, because this is a very clinical environment in addition to having to be in this costume PPE with the risk of becoming infected by mistake.",
"Moreover, participants were afraid of 'unknown territories', that is, they did not know the hospital ward, they were supposed to work in, and some team members had no recent experience of clinical work. One participant said: I didn't think these non-clinical people belonged in the team, because this is a very clinical environment in addition to having to be in this costume PPE with the risk of becoming infected by mistake. Those with non-clinical background were, however, aware of their limitations: I realized that I would not be the one in the front, I would not be managing patients directly. The importance ascribed to teamwork was evident in relation to fear. Participants described fear of working with people they had not worked with before: The weakest link in the preparation was that even though I knew their faces, I had never worked with them. Another issue was no-show by some team members in training sessions or in lectures: This is team-work, one does this and the other one does this, we help each other.",
"Participants described fear of working with people they had not worked with before: The weakest link in the preparation was that even though I knew their faces, I had never worked with them. Another issue was no-show by some team members in training sessions or in lectures: This is team-work, one does this and the other one does this, we help each other. Then you don't want to be working with someone who didn't show up. There were a lot of doctors who just dropped in, dropped out, and then dropped in again. I asked myself: Are these individuals … ready to take this on? Participants in the ETT mentioned the precautions they took or intended to take to cope with their feelings of fear, should Ebola emerge in Iceland.",
"I asked myself: Are these individuals … ready to take this on? Participants in the ETT mentioned the precautions they took or intended to take to cope with their feelings of fear, should Ebola emerge in Iceland. A major precaution was planning to avoid contact with the family while working with Ebola patients. One participant said: 'You thought … about your children at school … parents in the neighbourhood …' if they knew s he was working with an Ebola patient. For them, it was important they would have access to special accommodation in case of clinical EVD work 'so I wouldn't be exposing anyone or creating hysteria'. ETT members mentioned the extra insurance offered as a prerequisite for taking part in the team.",
"For them, it was important they would have access to special accommodation in case of clinical EVD work 'so I wouldn't be exposing anyone or creating hysteria'. ETT members mentioned the extra insurance offered as a prerequisite for taking part in the team. 'The normal insurance for LHS staff would not cover everything if we were to become sick or even lose our lives.' Amongst ER staff, the matter of insurance did seem to be less of an issue compared to the ETT. One respondent said: 'You are used to being at risk by many disease threats'. Furthermore, the issue of higher salaries and risk commission came up in the interviews, but overall did not matter as much to the participants as the insurance, or assurance of accommodation in case of need.",
"One respondent said: 'You are used to being at risk by many disease threats'. Furthermore, the issue of higher salaries and risk commission came up in the interviews, but overall did not matter as much to the participants as the insurance, or assurance of accommodation in case of need. Characteristics associated with Iceland and the Icelandic people were referred to repeatedly by participants. The concept 'Tiny Iceland' was often mentioned and emerged with positive and negative connotations. 'Tiny Iceland' referred to the size of the country and population and its perceived capability to still 'get the job done'. even though compromises had to be made.",
"'Tiny Iceland' referred to the size of the country and population and its perceived capability to still 'get the job done'. even though compromises had to be made. Comparing how Iceland handled its responsibilities differently from other countries of a larger size was often brought up, both with pride in Iceland as a strong independent nation, and with insecurities about its capacity in comparison to other countries. It was pointed out that since the preparedness process was in the hands of a few people, everyone knew their role. As one administrator said: This little hospital system, as complicated as it might seem every day, gives you the chance to just pick up the phone and call the one in charge. Being a small population presents challenges regarding resources, infrastructure, and specialized medical training to comply with standards of international actors.",
"As one administrator said: This little hospital system, as complicated as it might seem every day, gives you the chance to just pick up the phone and call the one in charge. Being a small population presents challenges regarding resources, infrastructure, and specialized medical training to comply with standards of international actors. Notions of Icelanders as resilient in spite of shortcomings were common; referring to the experience of preparedness planning and training, one health staff said: It was very much the Icelandic way, we'll manage, we'll work it out, and there was so much ingenuity. This notion of a particular Icelandic approach to coping, in spite of shortcomings, was also detected more generally, as in the statement: Would it have worked? Yes, it would have worked. Would it have been optimal?",
"Yes, it would have worked. Would it have been optimal? We cannot say, it would have been optimal; we can say, it would have been sufficient. In contrast to this, there were concerns about whether Icelandic aid workers falling ill in Ebolaaffected countries should be transferred to Iceland or to hospitals in other Nordic countries with better isolation units. Some of the participants trusted that patients with EVD would not be transferred to Iceland. One participant stated: You heard that Norwegians were criticized for transferring their aid worker from Africa to Norway. We don't know what would have happened if they would have transferred an Icelander into the country.",
"One participant stated: You heard that Norwegians were criticized for transferring their aid worker from Africa to Norway. We don't know what would have happened if they would have transferred an Icelander into the country. We don't have good enough isolation unitsyou are not supposed to send patients to a hospital that is less than 100%. I thought there was assurance in that. During the devastating Ebola epidemic in West Africa that spread to neighbouring sub-Saharan countries, North America, and Europe , preparedness plans were widely elaborated and later evaluated. Evaluations have, for example, been conducted in 11 African countries close to the epidemic , in the EU region , and the US .",
"During the devastating Ebola epidemic in West Africa that spread to neighbouring sub-Saharan countries, North America, and Europe , preparedness plans were widely elaborated and later evaluated. Evaluations have, for example, been conducted in 11 African countries close to the epidemic , in the EU region , and the US . Here we present data from a qualitative case study on the process, and experiences with establishing a preparedness plan for Ebola in Iceland in 2014. Interviews with staff who were engaged, either as administrators or frontline healthcare workers, alert us to the manner in which geographic, demographic, cultural, and organizational characteristics shaped the response. The results show that the process of establishing and training for preparedness was permeated by ambiguities of pride and pragmatism, trust, doubts, and fear. 'Getting the job done' theme 1 refers to the multitude of tasks and considerations that surrounds and feeds into the preparedness plan itself and are necessary for successful planning and implementation.",
"The results show that the process of establishing and training for preparedness was permeated by ambiguities of pride and pragmatism, trust, doubts, and fear. 'Getting the job done' theme 1 refers to the multitude of tasks and considerations that surrounds and feeds into the preparedness plan itself and are necessary for successful planning and implementation. Using the metaphors of 'hard core' and 'soft periphery', Langley and Denis emphasize the importance of relatively 'peripheral' concerns and processes for planning and implementation of new interventions. The hard core represents the actual intervention or goal, e.g. implementation of a preparedness plan. The soft periphery refers to all the contextually important networking, negotiations, and agreements necessary to deliver the hard core.",
"implementation of a preparedness plan. The soft periphery refers to all the contextually important networking, negotiations, and agreements necessary to deliver the hard core. If the soft periphery is neglected, it will cause multiple challenges in the implementation process, and the benefit of the hard core, the intervention itself, may not transpire as anticipated. Due attention to the soft periphery may, however, considerably promote the delivery of an innovation, and secure support from important stakeholders. In our data, one manager speaks of the preparedness process as dealing with a three-headed monster where every solution was followed by new problems. The data indicate that the process of dealing with 'the three headed monster' was given due attention as a means to successfully develop Iceland's preparedness plan.",
"In our data, one manager speaks of the preparedness process as dealing with a three-headed monster where every solution was followed by new problems. The data indicate that the process of dealing with 'the three headed monster' was given due attention as a means to successfully develop Iceland's preparedness plan. Comprehensive consultations and the involvement of many associated institutions were mentioned. Still ambiguity remained with some staff in terms of division of responsibilities and taskse.g. when transporting a patient potentially infected with Ebola from the airport to the hospital, and other such activities. During epidemics, rumours, gossip, and unreliable information on the news and social media spread rapidly, resulting in so-called 'infodemics' .",
"when transporting a patient potentially infected with Ebola from the airport to the hospital, and other such activities. During epidemics, rumours, gossip, and unreliable information on the news and social media spread rapidly, resulting in so-called 'infodemics' . The West African Ebola epidemic was covered widely by media , and the fear of Ebola reached every corner of the world, exemplified by travel bans from affected countries, and trade barriers , in contrast to the ongoing epidemic in the Democratic Republic of Congo . In our second theme, trust, doubt, and fear of health workers were represented. Although all intentions were good, concerns remained about the suitability and safety of the isolation ward, the PPE, and other tools, as well as adequate engagement of colleagues who might potentially work alongside them, in case an Ebola patient came to Iceland. The foreignness of putting on, removing, and working from within a PPE and the trustworthiness of available PPE were mentioned.",
"Although all intentions were good, concerns remained about the suitability and safety of the isolation ward, the PPE, and other tools, as well as adequate engagement of colleagues who might potentially work alongside them, in case an Ebola patient came to Iceland. The foreignness of putting on, removing, and working from within a PPE and the trustworthiness of available PPE were mentioned. In preparedness efforts in other countries, scarcity of resources in relation to manpower demand and problems with training and protocols involving PPE were common challenges . Similar problems were encountered in Iceland. Provisory treatment facility had to be designed, called 'camping site' by some, in contrast to facilities found elsewhere . Further, the ETT was established based on voluntary recruitment rather than on the staff's assigned roles within the healthcare system, a procedure that was deemed less than optimal.",
"Provisory treatment facility had to be designed, called 'camping site' by some, in contrast to facilities found elsewhere . Further, the ETT was established based on voluntary recruitment rather than on the staff's assigned roles within the healthcare system, a procedure that was deemed less than optimal. The members of the ETT pointed out that they had never worked together as a team under circumstances that demanded strict adherence to infectious control procedures. This eroded trust, compounded by the laissez-faire attitude of some of its members during the preparation exercises, possibly due to other competing tasks in a busy hospital and insufficient resources that hampered full participation . Further, it was a constraint that simulation exercises were not an option, found to be an important element in preparation for epidemics . This might have resulted in less than optimal staff protection for those who would have been in direct contact with an infected patient, as reported during the SARS epidemic in Canada .",
"Further, it was a constraint that simulation exercises were not an option, found to be an important element in preparation for epidemics . This might have resulted in less than optimal staff protection for those who would have been in direct contact with an infected patient, as reported during the SARS epidemic in Canada . Anthropological work on emergency preparedness emphasizes the connectedness between health professionals, technological devices, and knowledge as a prerequisite for successful preparedness. Wolf and Hall present preparedness efforts as a form of governance that involves human bodies those of health professionals , clinical architectures e.g. isolation wards , and technical artefacts gloves, protective suits, disinfectants, etc. .",
"isolation wards , and technical artefacts gloves, protective suits, disinfectants, etc. . During preparedness training and implementation, 'nursing bodies are transformed into instruments of preparedness', and become part of infrastructural arrangements. Health professionals are, here, both vulnerable and powerful tools in the management of contamination. The authors argue that successful planning, training, and implementation of a preparedness plan require such intrinsic connectedness. In the case of Ebola preparedness in Iceland, health professionals draw our attention to dilemmas of connectedness, and their assessment of the fact that these shortcomings might hamper the mobilization of 'preparedness within the human body'that is, the embodied experience, routine, and tacit knowledge which Wolf and Hall state are key to successful implementation.",
"The authors argue that successful planning, training, and implementation of a preparedness plan require such intrinsic connectedness. In the case of Ebola preparedness in Iceland, health professionals draw our attention to dilemmas of connectedness, and their assessment of the fact that these shortcomings might hamper the mobilization of 'preparedness within the human body'that is, the embodied experience, routine, and tacit knowledge which Wolf and Hall state are key to successful implementation. Repeated enactment of receiving and treating a patient with Ebola within experienced and trustful teams would probably enhance such embodiment, provided that there is justified trust in the involved technology. In addition, repetition would also strengthen the 'soft periphery' of preparedness, and divisions of responsibilities would be clearer manifested. In the third theme, we observe how notions of the 'Icelandic way' help participants make sense of ambiguities about Ebola preparedness. Loftsdóttir explored how people negotiated the imagination of the local and the global during the 2008 economic crisis in Iceland .",
"In the third theme, we observe how notions of the 'Icelandic way' help participants make sense of ambiguities about Ebola preparedness. Loftsdóttir explored how people negotiated the imagination of the local and the global during the 2008 economic crisis in Iceland . Notions of the intrinsic character of Iceland, and of being Icelandic, serve to underscore certain points and explain positive and negative experiences with the preparedness plan. Iceland is far away from the continents, but still connected through global needs for policy, risk of contamination, and dependency in terms of collaboration, in emergencies emerging from elsewhere. In our study, participants highlighted the importance of believing in oneself and the 'Icelandic way of doing things,' summed up in the paraphrase 'þetta reddast' things always have a way of working out in the end . The preparedness plan had to be completed, and adapted to Iceland's particular global situation.",
"In our study, participants highlighted the importance of believing in oneself and the 'Icelandic way of doing things,' summed up in the paraphrase 'þetta reddast' things always have a way of working out in the end . The preparedness plan had to be completed, and adapted to Iceland's particular global situation. In the 21st century, the world has faced new epidemic threats, such as SARS, and old scourges such as the plague have resurfaced . One of the main findings on Ebola preparedness measures in the EU was that measures taken were based on past preparedness and experience of other epidemics, such as SARS and H1N1 . Further, key stakeholders within each country found their measures to have been adequate for dealing with a single case of Ebola, as was the case in Iceland. A preparedness plan for pandemic influenzae in Iceland was elaborated in 2006activated in response to the H1N1 epidemic in 2009and revised in 2016 .",
"Further, key stakeholders within each country found their measures to have been adequate for dealing with a single case of Ebola, as was the case in Iceland. A preparedness plan for pandemic influenzae in Iceland was elaborated in 2006activated in response to the H1N1 epidemic in 2009and revised in 2016 . During the elaboration of these plans, communication among the different levels of the healthcare system and supporting agencies, such as the DCPEM, had been clearly defined, and proved to be useful in the preparedness for Ebola. Further, as found important in preparedness activities for pandemic influenzae elsewhere , honesty, transparency in communication, and sharing of information from managers to front-line health professionals, was found to be critical. It gave a feeling of being involved, and mitigated the fear that is so frequently encountered during epidemics . Iceland was far away from the epicentre of the Ebola epidemic in West Africa.",
"It gave a feeling of being involved, and mitigated the fear that is so frequently encountered during epidemics . Iceland was far away from the epicentre of the Ebola epidemic in West Africa. Yet this case study shows that health professionals felt the strain of possibly having to treat one or more patients with EVD. Their situation stands in sharp contrast to the situation in the three worst affected West African countries that lacked staff, stuff, space, and systems to effectively address the challenge of EVD. Although Icelandic health professionals had trust in the national healthcare system, and in their own capacity, doubt and fear influenced the reflections on preparedness planning of both administrators and healthcare staff. References to national identity and the characteristic of an 'Icelandic approach' to handling challenges assisted participants in coming to terms with the experienced shortcomings of the preparedness plan, and underscored the pride in the ingenuity applied in the process.",
"Although Icelandic health professionals had trust in the national healthcare system, and in their own capacity, doubt and fear influenced the reflections on preparedness planning of both administrators and healthcare staff. References to national identity and the characteristic of an 'Icelandic approach' to handling challenges assisted participants in coming to terms with the experienced shortcomings of the preparedness plan, and underscored the pride in the ingenuity applied in the process. These references negotiate the role and character of the nation of Iceland, and its role in a globalized world, as both a small and isolated nation on one hand, and a central and capable one, on the other. The experienced ambiguity needs attention in a health system and among healthcare staff that have to act resolutely and unfailingly, should they be placed in charge of containing contamination. This study points to the necessity of repeatedly re-enacting, as realistically as possible, the likely scenarios of receiving and treating one or more patients infected with Ebola or other contagious global health threats as a routine matter. This would assist in the identification of overlooked 'soft periphery' concerns, and promote embodied preparedness among teams of health care staff on the frontline.",
"This study points to the necessity of repeatedly re-enacting, as realistically as possible, the likely scenarios of receiving and treating one or more patients infected with Ebola or other contagious global health threats as a routine matter. This would assist in the identification of overlooked 'soft periphery' concerns, and promote embodied preparedness among teams of health care staff on the frontline. Geir Gunnlaugsson conceptualized the study, and took part in all necessary steps towards its completion, such as analysis and interpretation of data, and writing the manuscript for submission. Íris Eva Hauksdóttir collected and analysed the data as part of a master thesis work conducted under the supervision of all three co-authors, revised the manuscript, and approved the final version. Ib Bygbjerg took part in the interpretation of data, revision of the manuscript, and approved the final version. Britt Pinkowski Tersbøl took part in designing interview tools and in the thematic analysis of interview data, interpretation, revision of the manuscript, and approved the final version.",
"Ib Bygbjerg took part in the interpretation of data, revision of the manuscript, and approved the final version. Britt Pinkowski Tersbøl took part in designing interview tools and in the thematic analysis of interview data, interpretation, revision of the manuscript, and approved the final version. Dr. Gunnlaugsson reports he was the Chief Medical Officer CMO for Iceland, Directorate of Health, in the period 2010-2014. Other authors report no conflict of interest. The study was reported to the Data Protection Authority and approved by the National Bioethics Committee in Iceland number VSI- . Subsequently, the study was approved by the University Hospital Ethical Committee on 4 February 2016 number LSH .",
"The study was reported to the Data Protection Authority and approved by the National Bioethics Committee in Iceland number VSI- . Subsequently, the study was approved by the University Hospital Ethical Committee on 4 February 2016 number LSH . Participants signed an informed consent form before taking part in the study. Not applicable. The manuscript builds on the work of Íris Eva Hauksdóttir towards a MSc in Global Health, Section of Global Health, Department of Public Health, Copenhagen University, Denmark."
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What clinical condition is caused by Hantaan virus? | hemorrhagic fever with renal syndrome | [
"Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.",
"In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .",
"Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .",
"The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .",
". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .",
". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .",
"Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.",
"We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.",
"All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .",
"The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .",
": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.",
"critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .",
"The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .",
"Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .",
". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.",
"The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.",
"Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .",
"HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.",
"HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.",
"Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .",
"β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .",
"U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .",
"For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.",
"Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.",
"The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.",
"At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.",
"Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .",
"Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .",
"All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.",
"Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .",
"Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.",
"For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.",
"Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .",
"Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .",
"Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .",
"The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.",
"Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.",
"The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .",
"As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .",
"The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.",
"We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.",
"These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.",
", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.",
". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.",
"The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.",
"In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .",
"Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .",
". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .",
"Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.",
". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .",
"Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.",
"However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .",
". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.",
". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.",
". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.",
"We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .",
". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.",
"In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .",
"Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.",
"In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper."
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What is the structure of Hantaan virus? | enveloped, negative-sense RNA virus | [
"Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.",
"In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .",
"Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .",
"The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .",
". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .",
". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .",
"Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.",
"We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.",
"All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .",
"The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .",
": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.",
"critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .",
"The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .",
"Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .",
". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.",
"The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.",
"Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .",
"HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.",
"HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.",
"Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .",
"β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .",
"U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .",
"For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.",
"Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.",
"The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.",
"At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.",
"Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .",
"Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .",
"All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.",
"Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .",
"Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.",
"For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.",
"Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .",
"Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .",
"Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .",
"The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.",
"Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.",
"The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .",
"As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .",
"The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.",
"We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.",
"These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.",
", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.",
". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.",
"The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.",
"In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .",
"Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .",
". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .",
"Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.",
". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .",
"Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.",
"However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .",
". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.",
". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.",
". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.",
"We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .",
". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.",
"In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .",
"Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.",
"In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper."
] | 2,565 | 547 |
What the animal vector reservoir for Hantaan virus? | rodents | [
"Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.",
"In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .",
"Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .",
"The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .",
". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .",
". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .",
"Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.",
"We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.",
"All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .",
"The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .",
": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.",
"critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .",
"The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .",
"Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .",
". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.",
"The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.",
"Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .",
"HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.",
"HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.",
"Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .",
"β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .",
"U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .",
"For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.",
"Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.",
"The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.",
"At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.",
"Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .",
"Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .",
"All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.",
"Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .",
"Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.",
"For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.",
"Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .",
"Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .",
"Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .",
"The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.",
"Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.",
"The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .",
"As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .",
"The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.",
"We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.",
"These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.",
", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.",
". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.",
"The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.",
"In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .",
"Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .",
". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .",
"Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.",
". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .",
"Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.",
"However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .",
". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.",
". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.",
". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.",
"We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .",
". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.",
"In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .",
"Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.",
"In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper."
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What diagnostic test is correlated with the severity of HFRS? | plasma viral load | [
"Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.",
"In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .",
"Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .",
"The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .",
". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .",
". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .",
"Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.",
"We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.",
"All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .",
"The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .",
": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.",
"critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .",
"The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .",
"Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .",
". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.",
"The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.",
"Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .",
"HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.",
"HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.",
"Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .",
"β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .",
"U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .",
"For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.",
"Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.",
"The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.",
"At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.",
"Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .",
"Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .",
"All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.",
"Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .",
"Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.",
"For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.",
"Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .",
"Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .",
"Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .",
"The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.",
"Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.",
"The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .",
"As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .",
"The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.",
"We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.",
"These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.",
", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.",
". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.",
"The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.",
"In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .",
"Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .",
". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .",
"Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.",
". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .",
"Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.",
"However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .",
". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.",
". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.",
". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.",
"We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .",
". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.",
"In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .",
"Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.",
"In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper."
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How is Interferon used in practice? | an antiviral medicine | [
"Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.",
"In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .",
"Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .",
"The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .",
". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .",
". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .",
"Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.",
"We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.",
"All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .",
"The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .",
": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.",
"critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .",
"The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .",
"Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .",
". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.",
"The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.",
"Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .",
"HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.",
"HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.",
"Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .",
"β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .",
"U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .",
"For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.",
"Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.",
"The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.",
"At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.",
"Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .",
"Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .",
"All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.",
"Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .",
"Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.",
"For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.",
"Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .",
"Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .",
"Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .",
"The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.",
"Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.",
"The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .",
"As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .",
"The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.",
"We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.",
"These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.",
", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.",
". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.",
"The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.",
"In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .",
"Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .",
". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .",
"Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.",
". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .",
"Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.",
"However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .",
". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.",
". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.",
". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.",
"We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .",
". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.",
"In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .",
"Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.",
"In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper."
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What genotypes are associated with the severity of HFRS? | rs12252 C allele and CC genotype | [
"Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.",
"In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .",
"Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .",
"The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .",
". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .",
". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .",
"Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.",
"We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.",
"All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .",
"The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .",
": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.",
"critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .",
"The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .",
"Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .",
". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.",
"The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.",
"Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .",
"HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.",
"HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.",
"Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .",
"β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .",
"U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .",
"For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.",
"Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.",
"The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.",
"At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.",
"Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .",
"Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .",
"All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.",
"Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .",
"Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.",
"For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.",
"Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .",
"Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .",
"Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .",
"The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.",
"Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.",
"The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .",
"As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .",
"The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.",
"We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.",
"These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.",
", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.",
". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.",
"The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.",
"In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .",
"Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .",
". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .",
"Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.",
". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .",
"Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.",
"However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .",
". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.",
". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.",
". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.",
"We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .",
". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.",
"In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .",
"Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.",
"In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper."
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Which IFITM proteins have been shown to possess antiviral activity? | IFITM1, 2, and 3 | [
"Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.",
"In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .",
"Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .",
"The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .",
". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .",
". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .",
"Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.",
"We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.",
"All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .",
"The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .",
": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.",
"critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .",
"The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .",
"Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .",
". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.",
"The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.",
"Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .",
"HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.",
"HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.",
"Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .",
"β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .",
"U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .",
"For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.",
"Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.",
"The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.",
"At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.",
"Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .",
"Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .",
"All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.",
"Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .",
"Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.",
"For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.",
"Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .",
"Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .",
"Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .",
"The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.",
"Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.",
"The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .",
"As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .",
"The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.",
"We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.",
"These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.",
", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.",
". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.",
"The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.",
"In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .",
"Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .",
". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .",
"Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.",
". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .",
"Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.",
"However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .",
". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.",
". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.",
". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.",
"We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .",
". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.",
"In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .",
"Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.",
"In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper."
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What is the hypothesized mechanism by which IFITMs work? | restrict viral infection at the stage of cellular entry | [
"Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.",
"In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .",
"Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .",
"The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .",
". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .",
". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .",
"Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.",
"We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.",
"All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .",
"The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .",
": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.",
"critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .",
"The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .",
"Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .",
". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.",
"The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.",
"Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .",
"HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.",
"HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.",
"Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .",
"β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .",
"U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .",
"For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.",
"Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.",
"The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.",
"At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.",
"Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .",
"Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .",
"All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.",
"Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .",
"Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.",
"For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.",
"Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .",
"Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .",
"Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .",
"The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.",
"Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.",
"The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .",
"As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .",
"The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.",
"We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.",
"These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.",
", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.",
". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.",
"The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.",
"In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .",
"Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .",
". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .",
"Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.",
". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .",
"Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.",
"However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .",
". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.",
". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.",
". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.",
"We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .",
". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.",
"In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .",
"Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.",
"In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper."
] | 2,565 | 555 |
What genotype causes truncation of the IFITM3 protein? | rs12252 C allele | [
"Hantaan virus HTNV causes hemorrhagic fever with renal syndrome HFRS . Previous studies have identified interferon-induced transmembrane proteins IFITMs as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms SNP rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay.",
"In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response NRIR . Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS .",
"Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS . . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . .",
"The human family of interferon-induced transmembrane proteins IFITMs was discovered 25 years ago to consist of interferon-stimulated genes ISGs . . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity . . Different IFITM proteins have different antiviral spectrum . . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice . , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus . . . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . .",
". . . . . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells . . Single nucleotide polymorphisms SNPs are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro . . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus . . HTNV has been shown to induce a type I interferon response though in later time postinfection . .",
". HTNV has been shown to induce a type I interferon response though in later time postinfection . . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection . , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity . . Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . .",
"Among them, negative regulator of interferon response NRIR lncRNA NRIR, also known as lncRNA-CMPK2 is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection . . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection.",
"We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained.",
"All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows .",
"The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay ELISA in our department. The classification of HFRS severity and the exclusion criteria were described as follows . : white blood cells WBC , platelets PLT , blood urea nitrogen BUN , serum creatinine Scr , and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory shown in Table 1 . According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described . : . mild patients were identified with mild renal failure without an obvious oliguric stage; .",
": . mild patients were identified with mild renal failure without an obvious oliguric stage; . moderate patients were those with obvious symptoms of uremia, effusion bulbar conjunctiva , hemorrhage skin and mucous membrane , and renal failure with a typical oliguric stage; . severe patients had severe uremia, effusion bulbar conjunctiva and either peritoneum or pleura , hemorrhage skin and mucous membrane , and renal failure with oliguria urine output, 50-500 ml/day for ≤5 days or anuria urine output, <50 ml/day for ≤2 days; and . critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group.",
"critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria urine output, 50-500 ml/day for >5 days, anuria urine output, <50 ml/day for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, .",
"The exclusion criteria for this study were patients with: . any other kidney disease, . diabetes mellitus, . autoimmune disease, . hematological disease, . cardiovascular disease, . viral hepatitis types A, B, C, D, or E , or . any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period . . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ .",
"Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit Gentra Systems, Minneapolis, MN, USA . The region encompassing the human IFITM3 rs12252 were amplified by PCR forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′ . The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer Thermo Scientific, Waltham, MA, USA . The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project The HTNV load in plasma samples collected during the acute phase from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods . . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA .",
". Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits Invitrogen, Carlsbad, CA, USA . The SuperScript III Platinum One-Step Quantitative RT-PCR System kit Invitrogen, Carlsbad, CA, USA was employed for the real-time RT-PCR assay. The primers and probe provided by Sangon Biotech, Shanghai, China were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′- FAM ATCCCTCACCTTCTGCCTGGCTATC TAMRA -3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators.",
"The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler Bio-Rad, Hercules, CA, USA with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator.",
"Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells ScienCell Research Laboratories, Carlsbad, CA, USA were grown in ECM BulletKit ScienCell Research Laboratories, Carlsbad, CA, USA in a 5% CO2 incubator. A549 cells ATCC Cat# CRM-CCL-185, RRID:CVCL_0023 were grown in our laboratory in DMEM with 10% FBS Thermo Scientific, Waltham, MA, USA in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . .",
"HTNV strain 76-118 was cultured in Vero E6 cells ATCC Cat# CRL-1586, RRID:CVCL_0574 in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein NP as previously described . . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source Piscataway, NJ, USA and dissolved in the buffer provided by the manufacturer composition not disclosed . HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture.",
"HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 purchased from GENECHEM, Shanghai, China at a moi of 10. Puromycin 2 μg/ ml for HUVEC and 6 μg/ml for A549 cells was used to create cell lines stably expressing IFITMs. Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed.",
"Cells were transfected with control scrambled short interfering RNA siRNA , IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA 10 nM using Lipofectamine 3000 transfection reagent Invitrogen, Carlsbad, CA, USA . SiRNAs were purchased from Origene Rockville, MD, USA , and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent Invitrogen, Carlsbad, CA, USA , and cDNA was synthesized using the K1622 kit Thermo Scientific, Waltham, MA, USA . Quantitative realtime PCR qPCR was performed using SYBR Premix Ex Taq II Takara Biotechnology Co., Dalian, China with a Bio-Rad iQ5 cycler Bio-Rad, Hercules, CA, USA . β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ .",
"β-actin was used as the reference gene. The primers Sangon Biotech, Shanghai, China were as follows: IFITM1 forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′ ; IFITM2 forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′ ; IFITM3 forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′ ; IFITM3 pre-mRNA forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′ ; HTNV S segment forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′ ; β-actin forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′ ; NRIR forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′ ; NRAV forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′ . For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit Invitrogen, Carlsbad, CA, USA with a specific primer in gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . MiR-130a level was determined using the gene-specific TaqMan assay kit 000454, Invitrogen, Carlsbad, CA, USA . U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . .",
"U6 001973, Invitrogen, Carlsbad, CA, USA was used as an endogenous control . . Because the pre-mRNA levels can represent the initial transcription rate . , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described . . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . .",
"For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon . . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment 20 IU/ml for 12 h after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay RIPA buffer Sigma-Aldrich, St. Louis, MO, USA . Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C.",
"Equal amounts of protein 20 μg protein/lane were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane Millipore, Billerica, MA, USA . After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405 , IFITM2, IFITM3 Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821 , and β-actin Proteintech, Wuhan, Hubei, China or HTNV NP provided by the Department of Microbiology, The Fourth Military Medical University overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody Cell Signaling Technology, Danvers, MA, USA for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit Millipore, Billerica, MA, USA and visualized using X-ray film. The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS.",
"The blot densities were analyzed using the Quantity One software Bio-Rad, Hercules, CA, USA . In addition, the RIPA buffer contains 50mM Tris pH = 7.4 , 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail Roche, Basel, Switzerland was added before use. The cells were cultured on glass coverslips Millipore, Billerica, MA, USA until they were semi-confluence and then incubated with HTNV for 60 min moi = 1 . At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images.",
"At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 Sigma-Aldrich, St. Louis, MO, USA , and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546 , IFITM3, lysosome-associated membrane glycoprotein 1 LAMP1, Cell Signaling Technology, Danvers, MA, USA , or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 Abcam, Cambridge, MA, USA secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system Olympus, Tokyo, Japan were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry.",
"Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K 0.1 mg/ml, Thermo Scientific, Waltham, MA, USA . To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl .",
"Adsorption of HTNV moi = 1 was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium 20 mM sodium succinate, pH = 5.5 for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl . . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA .",
"All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 GraphPad Software, La Jolla, CA, USA . For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant.",
"Differences among groups were determined by one-way analysis of variance ANOVA with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 .",
"Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section \"Material and Methods. \" We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database 68.29 vs. 52.16%, P = 0.0076 . The frequency of rs12252 C in severe patients was also higher than those mild patients 68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 . These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio 95% CI of 2.124 1.067-4.230 . For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity.",
"For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database 26.92% CC genotype, P = 0.03 as well as mildly infected patients 14.29%, P = 0.02, Figures 1A,B ; Table 2 . However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. c The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population.",
"Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase Figure 1C . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . .",
"Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes leads to an impaired anti-influenza activity . . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated NΔ21 proteins Figure 2A with c-myc-tag to HUVEC and A549 cell using lentivirus vectors Figure 2B . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment Figure 2C and more positive of HTNV NP Figure S3 in Supplementary Material . Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material .",
"Indeed, compared with the mock empty vector -infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells Figures 2C,D ; Figure S3 in Supplementary Material . To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells Figures 3A,B ; Figure S1 in Supplementary Material . While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h .",
"The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs Figure S2 in Supplementary Material , and the effect of the best oligo against each IFITMs IFITM1C, IFITM2A, IFITM3B was tested by Western blot in A549 Figure 4A and HUVEC cells Figure 4B . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment 20 IU/ml for another 12 h . The cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection.",
"Knockdown of IFITM1 also partially restored the HTNV level in A549 cells Figures 4C,D . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells Figure 5A , and the cells were then challenged with HTNV moi = 1 for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells Figures 5B-D . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells Figures 5B-D . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results.",
"The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP Figure S3 in Supplementary Material . These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV moi = 1 at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B .",
"As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section \"Materials and Methods.\" Expression of IFITM3 did not affect HTNV binding Figure 6A but significantly suppressed HTNV entry in both HUVEC and A549 cells Figure 6B . iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy Figure 6C . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . .",
"The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells . . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry . , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells.",
"We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one downregulated after HTNV infection Figure 7A ; Figure S4 in Supplementary Material in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector Figure 7B . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells Figures 7C-E . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae.",
"These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome HPS in humans . . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury . , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment.",
", causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS .. However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response . . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified.",
". However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity.",
"The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS.",
"In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication . . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . .",
"Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals . . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families including filoviruses, rhabdoviruses, and flaviviruses 7, . . . 30 . For example, HIV-1 and HCV infection are inhibited by IFITM1 . . . . . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry . . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . .",
". Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein . . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles . , whereas IFITM3 confines influenza virus in acidified endosomal compartments . . Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . .",
"Notably, retrovirus subvirus particles ISVPs , which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry . . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging FLIM suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes . . In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells.",
". In the present study, we demonstrated that IFN-α2a 20 U/ ml significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . .",
"Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV . . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells.",
"However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices shown in Figure 2A as black boxes in IFITM3 . . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line .",
". There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids deleted part, shown in Figure 2A as red dotted line . Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence . . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells . . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro.",
". We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed . . In Chinese patients infected with influenza A H1N1 virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 . . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients.",
". In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity.",
"We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression . . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . .",
". lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes . . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection . . Mir-130a was also reported as a regulator of IFITM1 . . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection.",
"In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material .",
"Another lncRNA that can regulate IFITM3, i.e., NRAV NR_038854 , remained unchanged after HTNV infection Figures S4A,B in Supplementary Material . Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection Figures S4C,D in Supplementary Material . In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease.",
"In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein NΔ21 that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper."
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What is a Hantavirus? | zoonotic diseases | [
"Hantavirus infection, which causes zoonotic diseases with a high mortality rate in humans, has long been a global public health concern. Over the past decades, accumulating evidence suggests that long noncoding RNAs lncRNAs play key regulatory roles in innate immunity. However, the involvement of host lncRNAs in hantaviral control remains uncharacterized. In this study, we identified the lncRNA NEAT1 as a vital antiviral modulator. NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection.",
"NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection. Further investigation suggested that NEAT1 served as positive feedback for RIG-I signaling. HTNV infection activated NEAT1 transcription through the RIG-I–IRF7 pathway, whereas NEAT1 removed the transcriptional inhibitory effects of the splicing factor proline- and glutamine-rich protein SFPQ by relocating SFPQ to paraspeckles, thus promoting the expression of RIG-I and DDX60. RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections.",
"RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections. IMPORTANCE Hantaviruses have attracted worldwide attention as archetypal emerging pathogens. Recently, increasing evidence has highlighted long noncoding RNAs lncRNAs as key regulators of innate immunity; however, their roles in hantavirus infection remain unknown. In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling.",
"In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling. This lncRNA was induced by HTNV through the RIG-I–IRF7 pathway in a time- and dose-dependent manner and promoted HTNV-induced IFN production by facilitating RIG-I and DDX60 expression. Intriguingly, NEAT1 relocated SFPQ and formed paraspeckles after HTNV infection, which might reverse inhibitive effects of SFPQ on the transcription of RIG-I and DDX60. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections.",
"To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections. Text: glycoprotein GP , and viral RNA-dependent polymerase protein RdRp , respectively. Humans become infected by inhaling contaminated aerosols or by coming into contact with rodent excreta, and they develop two severe acute diseases, namely, hemorrhagic fever with renal syndrome HFRS and hantavirus pulmonary syndrome HPS . . Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . .",
". Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . . Chinese HFRS cases, mainly caused by Hantaan virus HTNV infection, account for approximately 90% of all global cases, with a mortality rate ranging from 0.1 to 15% . . Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions.",
"Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions. Cellular pathogen recognition receptors PRRs , including Toll-like receptors TLRs and RIG-I like receptors RLRs , can detect distinct pathogen-associated molecular patterns PAMPs and trigger the expression of IFNs and cytokines. RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression.",
"RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression. lncRNA nuclear paraspeckle assembly transcript 1 NEAT1 is an essential architectural constituent of paraspeckles in the mammalian nucleus, interacting with Drosophila DBHS RNA-binding proteins such as the splicing factor proline-and glutamine-rich protein SFPQ and the non-POU domain-containing, octamer-binding protein NONO/p54 . . To date, two isoform transcripts of the NEAT1 gene have been identified, namely, the 3.7-kb NEAT1-1 MEN and the 23-kb NEAT1-2 MEN Fig. 1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . .",
"1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . . , promoting cancer formation in mice by dampening oncogene-dependent activation of p53 . . Nevertheless, studies assessing the function of NEAT1 in viral infections are scarce. Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication.",
"Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication. Further investigation showed that NEAT1 promoted RIG-I and DDX60 expression by relocating SFPQ and removing the transcriptional inhibitory effects of SFPQ, which are critical for IFN responses against HTNV infection. We also found that RIG-I signaling, rather than TLR3 and TLR4, accounted for the elevation of HTNV-induced NEAT1. Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection.",
"Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection. As shown in Fig. 1B , the NEAT1 level in the HTNV group was higher than that in the mock group P ϭ 6.86 ϫ 10 Ϫ13 , false discovery rate FDR ϭ 9.75 ϫ 10 Ϫ12 or the 60 Co-inactivated HTNV group P ϭ 1.75 ϫ 10 Ϫ14 , FDR ϭ 3.10 ϫ 10 Ϫ13 ; however, the difference between the 60 Co-inactivated HTNV group and the mock group was not significant P ϭ 0.21034, FDR ϭ 0.58211 . To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig.",
"To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig. 1A , were applied to quantify NEAT1 RNA isoforms by quantitative real-time PCR qRT-PCR . It has been reported that NEAT1-2 rather than NEAT1-1 plays a key regulatory role in paraspeckle formation . , and we also found that elevated NEAT1 levels depend on live HTNV infection rather than 60 Co-inactivated HTNV stimulation Fig. 1C . Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D .",
"Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D . To further investigate whether NEAT1 expression was altered in other cell lines, HEK293, HeLa, and A549 cells were used. All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G .",
"All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G . Similar to the data obtained from HUVECs, NEAT1 was indeed upregulated by HTNV at a multiplicity of infection MOI of 1 beginning at 24 hpi in HUVECs and A549, HEK293, and HeLa cells, and the increasing tendency occurred in a time-dependent manner Fig. 1H . Of note, the NEAT1 elevation at 2 hpi might have been unrelated to the virus but resulted in cellular stress responses. Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I .",
"Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I . NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. The abovedescribed data showed that HTNV infection increased NEAT1, and we wondered how NEAT1 could reciprocally influence HTNV replication. The small interfering RNA siRNA transfection efficiency in HUVECs was confirmed by flow cytometry, and NEAT1 expression was significantly decreased, as assessed by qRT-PCR after RNA interference RNAi Fig. 2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2.",
"2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2. Compared with the cells transfected with control siRNA negative control NC , HUVECs with si-NEAT1 could dramatically promote HTNV NP production, and NP expression seemed to be related to the amount of applied si-NEAT1 Fig. 2B . Intriguingly, depletion of NEAT1-2 alone could mimic the antiviral effects of simultaneous NEAT1-1 and NEAT1-2 silencing Fig. 2C , indicating that NEAT1-2 was critical for the antiviral responses. Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing.",
"Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing. On the other hand, plasmids, of which pCMV-NEAT1-1 is transcribed into the 3.7-kb NEAT1-1 MEN and pCMV-NEAT1-2 is transcribed into the 2-to 3-kb NEAT1-2 MEN , were applied to directly investigate the role of NEAT1 in HTNV infection Fig. 2F . Surprisingly, we found NEAT1-1 overexpression restricted NEAT1-2 transcription Fig. 2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G .",
"2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G . Furthermore, overexpression of NEAT1-2 instead of NEAT1-1 could efficiently suppress HTNV replication Fig. 2H . NEAT1-1 upregulation even aggravated HTNV infection Fig. 2H , which may be the result of downregulation of NEAT1-2. Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig.",
"Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig. 2J were limited in HUVECs in which NEAT1-2 was ectopically expressed in comparison to those transfected with control vector or pCMV-NEAT1-1. These data further showed that NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. Alteration of NEAT1-2 affects HTNV-induced IFN expression in HUVECs. IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . .",
"IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . . It has been reported that the GnT of hantaviruses suppressed IFN- expression of host cells at an early stage of infection . . Here, we also found that HUVECs could not efficiently produce IFN- until 12 hpi at an MOI of 0.1 or until 24 hpi at an MOI of 1 Fig. 3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig.",
"3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig. 3B , suggesting that NEAT1-2 expression increased just before IFN production. We found that expression of endogenous IFN- mRNA was much lower in cells transfected with si-NEAT1-2 at MOIs of both 0.1 Fig. 3C and 1 Fig. 3D than in those transfected with control siRNA NC . In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E .",
"In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E . More importantly, HUVECs transfected with pCMV-NEAT1-2 conspicuously increased IFN- gene expression compared with those cells with vector plasmids at 12 hpi MOI ϭ 1 , demonstrating that NEAT1-2 overexpression accelerated robust IFN responses in host cells against HTNV infection. With a dual luciferase reporter system Twenty-four hours after transfection, the cells expressing FAM were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1.",
"Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the NC group . NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g .",
"NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 0.1 for 48 h. The expression of HTNV NP was measured by Western blotting. C HUVECs were treated as described for panel A, right, but at an MOI of 0.1. In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.",
"In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NC group . D HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group .",
"The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . E HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g .",
"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g . Twenty-four hours after transfection, the cells expressing green fluorescent protein GFP were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with control plasmids vector , pCMV-NEAT1-1, or pCMV-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig.",
"Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig. 3F . These results showed that NEAT1-2 regulated HTNV-induced IFN- expression. To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig.",
"To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig. 3G . In addition, in cells with high NEAT1-2 expression, treatment with neutralizing antibodies NAbs of IFN-␣ and IFN- could counteract the antiviral effects of NEAT1-2 MOI ϭ 1 , and the compensatory effects were dependent on the magnitude of the NAbs. Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production.",
"Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production. PRRs maintain a vital role in the promotion of IFN responses, and we conjectured that NEAT1 might amplify IFN responses by modulating these molecules. TLR3, TLR4, and RIG-I have been shown to recognize HTNV infection . . DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate .",
". DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate . , Here, we found that multiple Toll-like receptors like TLR1, TLR2, TLR3, and TLR4, as well as MDA5, were increased after HTNV infection, but none of them were influenced by silencing NEAT1-2 Fig. 4A . The upregulated RIG-I and DDX60 were blocked in the cells with low NEAT1-2 expression after HTNV infection Fig. 4A . HUVECs with declining NEAT1-2 expression showed gradually decreasing expression of RIG-I and DDX60 Fig. 4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C .",
"4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C . These data indicated that NEAT1-2 could positively modulate RIG-I and DDX60 expression, while the role of RIG-I and DDX60 upon HTNV infection is obscure. We then found that RIG-I and DDX60 colocalized after HTNV infection Fig. 4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown .",
"4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown . Simultaneously knocking down RIG-I and DDX60 significantly promoted HTNV NP expression Fig. 4E , and knockdown of both of them could greatly affect IFN- expression Fig. 4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig.",
"4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig. 4H , indicating that efficient anti-HTNV responses might depend on the interactive effects of DDX60 and RIG-I. More importantly, RIG-I or/and DDX60 overexpression enhanced HTNV-induced IFN- expression, and they had synergistic effects on IFN- production Fig. 4I and J . Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60.",
"Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60. NEAT1 was found to interact with SFPQ by RNA immunoprecipitation RIP after HTNV infection Fig. 5A , indicating that modulatory effects of NEAT1 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively .",
"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1 for 48 h. The expression of HTNV NP was measured by Western blotting. H HUVECs were treated as described for panel F, right. In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.",
"In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . H HUVECs were treated as described for panel F, right. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group .",
"The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . I HUVECs were treated as described for panel F, right. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ.",
"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ. Interestingly, the protein level of SFPQ, as well as another paraspeckle-forming constituent, NONO, remained unchanged after HTNV infection Fig. 5B or after NEAT1 overexpression and knockdown Fig. 5C . However, SFPQ became centralized rather than diffuse in the nucleus after HTNV infection Fig. 5D . The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig.",
"The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig. 5F and G , which might have been related to the increase in RIG-I Fig. 5H and DDX60 Fig. 5I . SFPQ has been suggested to bind to the promoter region of RIG-I and DDX60 ., thus preventing the expression of RIG-I and DDX60. Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription.",
"Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription. Interestingly, overexpression of the S or M segment of HTNV in HEK293 cells failed to induce NEAT1 expression, suggesting that NEAT1 transcription was closely related to live viral replication Fig. 6A . Of note, the upregulation of NEAT1 by HTNV could not be reversed by applying IFN-I neutralizing antibodies Fig. 6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig.",
"6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig. 6C, D, and E or cytokines Fig. 6F and G . We conjectured that NEAT1 expression was related to the activation of PRRs. By knocking down several PRRs, we found that the RIG-I and TLR4 pathways played important roles in HTNV-induced NEAT1 upregulation Fig. 7A . Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C .",
"Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C . Moreover, using STAT1 as a positive control, we found that the transcription factor IRF7, rather than IRF3 and p65, translocated into the nucleus in HTNV-infected HUVECs at 2 dpi Fig. 7D . Furthermore, IRF7 knockdown blocked HTNV-induced NEAT1 upregulation Fig. 7E . Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice.",
"Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice. Although cell-based experiments revealed that NEAT1-2 is a crucial regulator of innate antihantaviral responses, its function in vivo has remained unclear. To address this question, we intravenously injected siRNAs targeting mouse NEAT1-2 at 1 day before HTNV infection. NEAT1-2 expression levels in the liver, kidney, and spleen were reduced at 2 dpi Fig. 8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions.",
"8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions. Body weight loss in NEAT1-2-depleted mice was observed from 2 dpi to 5 dpi, and the IFN production in serum was remarkably decreased in the NEAT1-2 silenced group than those in the NC group at 3 dpi Fig. 8B . As expected, NEAT1-2 knockdown mice showed considerably higher HTNV NP levels in the liver, spleen, and kidney at 3 dpi Fig. 8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D .",
"8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D . In addition, reduced inflammatory cell filtration but increased tissue injury was found in NEAT1-2 knockdown mice during the early stage of infection Fig. 8E . Infiltration of macrophages in the spleen was attenuated Fig. 8F , and the activation of macrophages was also suppressed by flow cytometry; data not shown . Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G .",
"Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G . Nevertheless, NEAT1-2 silencing had no effect on the production of neutralizing antibodies at 7 dpi data not shown . The above-described findings indicated that NEAT1-2 depletion might influence multiple aspects of the innate immune response in HTNV-infected mice. Innate immunity is a phylogenetically ancient and conserved system that counteracts invading microbes, the regulatory mechanism of which is sophisticated and complex. Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . .",
"Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . . In this report, we first demonstrated that NEAT1 was induced by HTNV through the RIG-I-IRF7 pathway and served as positive feedback for RIG-I signaling. Using DGE analysis, we observed upregulated NEAT1 and confirmed its alteration in To further determine the function of NEAT1 after HTNV infection in vivo, mice were injected intravenously with si-NEAT1-2 1 g/g or nontarget control siRNA NC 1 g/g ; 1 day later, they were infected with HTNV 100 LD 50 by intramuscular injection. A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group .",
"A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group . B The effects of NEAT1 on HTNV virulence in mice were determined by body weight loss from 0 to 10 dpi left panel, n ϭ 10 in each group . The IFN- in sera of different groups was measured by ELISA at 3dpi right panel, n ϭ 8 in each group . Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi.",
"Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi. Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . D NEAT1 effects on HTNV infection kinetics at 3 dpi were determined by testing the HTNV titers in livers, spleens, and kidneys. Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group .",
"Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group . After HTNV infection, livers in the NC group showed inflammatory cell infiltration in certain regions, while those in the si-NEAT1-2 group showed slight acute viral hepatitis. Spleens in the NC group showed lymph node hyperplasia, while those in the si-NEAT1-2 group were severely congestive. Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented.",
"Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented. G CD3 ϩ CD8 ϩ IFN-␥ ϩ T cells were analyzed by flow cytometry at 3 dpi, and the results obtained for three mice in each group are presented. different cell lines. To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression.",
"To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression. By virtue of RNAi, the RIG-I-IRF7 pathway was confirmed to be necessary for HTNV-triggered NEAT1 elevation. Recently, large-scale transcriptomic studies identified numerous noncoding transcripts in the mammalian genome, which were speculated to influence diverse biological processes. Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression .",
"Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression . . TLR2 activation or tumor necrosis factor alpha TNF-␣ stimulation induces transcription of the lncRNA THRIL, the downregulation of which impairs TNF-␣ and IL-6 secretion . . TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . .",
". TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . . The lncRNA Lethe, triggered by TNF-␣ and IL-1, acts as a negative feedback regulator of NF-B signaling . . The roles of lncRNAs in host-virus interactions have been progressively unveiled. Various viruses, such as influenza virus IAV , coronavirus, enterovirus, human immunodeficiency virus HIV , hepatitis B virus HBV , hepatitis C virus HCV , Japanese encephalitis virus JEV , and rabies virus, have been reported to activate the transcription of different lncRNAs in host cells 11, . . . .",
". . . Importantly, multiple lncRNAs have been shown to affect the IFN response in recent years and have gradually become hot spots in the field of antiviral research. NeST was shown to enhance IFN-␥ production, controlling the susceptibility of mice to persistent Theiler's virus infection as well as resistance to Salmonella enterica serovar Typhimurium infection . . Both CMPK2 and NRAV were identified as negative regulators of IFN immune reactions. CMPK2, induced by IFN-␣ or HCV infection, suppresses various ISGs, the knockdown of which dramatically blocks HCV replication . . NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . .",
". NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . . Numerous lncRNAs, including lnc-ISG15 and ISR2, respond to IFNs such as ISGs, although their actual function requires further investigation . . Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection.",
"Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection. NEAT1 has been reported to interact with Drosophila DBHS RNA-binding proteins e.g., SFPQ, NONO-p54nrb, and PSPC1 , recruiting them to paraspeckles, a nuclear substructure found in all cultured and primary cells except embryonic stem cells . . The versatile function of NEAT1 is rapidly progressing in multiple areas of biology. NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . .",
"NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . . NEAT1 also participates in neurodegenerative diseases such as Huntington's disease . and seems to potentially contribute to the elevated production of a number of cytokines and chemokines in patients with systemic lupus erythematosus SLE . . Furthermore, poly I·C can activate NEAT1 transcription through the TLR3 pathway, whereas NEAT1 positively regulates IL-8 transcription and potentially affects the expression of multiple ISGs after poly I·C stimulation . . In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . .",
"In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . . However, the role of NEAT1 in hantaviral infection remains unclear. In this report, NEAT1 has been identified as an important regulator of the host innate immune system against HTNV infection. Elevated NEAT1 promotes IFN secretion, most likely by enhancing RIG-I and DDX60 expression. DDX60, a DEXD/H box RNA helicase similar to Saccharomyces cerevisiae Ski2, is induced after viral infection . . DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response.",
". DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response. However, the antiviral effects of DDX60 seem to vary among viruses . . We found that NEAT1-regulated DDX60 was involved in IFN production in response to HTNV infection. In HTNV-infected cells, double-stranded RNA dsRNA could not be detected, and it is unclear how host PRRs, especially RIG-I, recognize HTNV invasion . . Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation.",
". Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation. Of note, we applied multiple cell lines to explore the role of NEAT1 during HTNV infection. HTNV primarily targets vascular endothelial cells in vivo and contributes to the increased vascular permeability and coagulation disorders in HFRS; hence, HUVECs are the most common in vitro cell model to study host innate immunity against HTNV infection or viral pathogenesis . . EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies .",
". EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies . . A549 cells were once used to isolate HTNV, and they were confirmed to be a mature model of infection . . . . . Additionally, Huh 7.0 and Huh 7.5 RIG-I Ϫ cells used in our study have been reported to be infected by HTNV by Lee et al. . and can be used as a cell model to study immune responses against HTNV replication . . Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV.",
". Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV. Using qRT-PCR, Western blotting, and immunofluorescence assays, we have also shown that both HEK293 and HeLa cells can be infected by HTNV. To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production.",
"To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production. Alterations in the relative fluorescence intensity of NP after silencing or overexpressing NEAT1-2 did not seem to be as remarkable as qRT-PCR or Western blot analysis results. The NP spotted and exhibited in the ICW results forms obvious stains that mimic PFU. However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays.",
"However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays. The RNAi studies in vivo are encouraging Fig. 8 , but the NC used by our group was not mutated si-NEAT1-2 i.e., same sense strand, but with a point mutation in the targeting strand . The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling.",
"The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling. After observing that NEAT1 can regulate IFN expression by HTNV infection, we were interested in the function of NEAT1. We noticed that silencing NEAT1-2 or ectopically expressing NEAT1-2 could not inhibit or enhance IFN expression without HTNV infection Fig. 3F , which indicated that NEAT1-2 could not directly affect IFN- expression. This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression.",
"This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression. Recent findings have shown that RIG-I signaling is essential for an efficient polyfunctional T cell response during IAV infection . . Indeed, we found that the function of T cells was suppressed after NEAT1-2 depletion in our animal experiments Fig. 8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection.",
"8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection. HTNV infection induced NEAT1 expression through the RIG-I-IRF7 pathway, while NEAT1 displayed positive feedback for RIG-I signaling. NEAT1 relocated SFPQ from the potential promoter region of several antiviral genes to the paraspeckles, removing the transcriptional inhibitory effects of SFPQ. This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 .",
"This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 . nontarget control i.e., negative control NC or targeted RIG-I and DDX60 were designed by Gene-Pharma as follows: NC, 5=- UUCUUCGAACGUGUCACGUTT-3=; si-RIG-I-1, 5=-GCCCAUUUAAACCAAGAAATT-3=, si-RIG-I-2, 5=-GGUGGAGGAUAUUUGAACUTT-3=, and si-RIG-I-3, 5=-CCCAACGAUAUCAUUUCUTT-3=; si-DDX60-1, 5=-GUCCAGGUGUCAGUUUGAUTT-3=, si-DDX60-2, 5=-CCGAAGUGAAGAAGGUAAATT-3=, and si-DDX60-3, 5=-GAUGGAUGCUAGGAAAUAUTT-3=. The pCMV-NEAT1-1 and pCMV-NEAT1-2 plasmids, which transcribe NEAT1-1 and NEAT1-2, respectively, were provided by Nakagawa Shinichi . . The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . .",
". The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . . Abs against RIG-I, IRF3, IRF7, p65, and STAT1, as well as the neutralizing antibodies against IFN-␣ and IFN-, were purchased from Abcam Cambridge, MA, USA . Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China .",
"Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China . The Abs targeting CD11b, F4/80, CD3, CD8, IFN-␥, inducible nitric oxide synthase iNOS , and CD206 for flow cytometry were purchased from BD Biosciences San Jose, CA, USA . IFN-␣, -, and -␥, TNF-␣, and IL-1 were from PeperoTech Rocky Hill, NJ . ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis.",
"ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis. Total cellular RNAs were extracted with RNAiso TaKaRa, Dalian, China , the concentration of which was measured using a NanoDrop 1000 spectrophotometer. Reverse transcription RT was then performed with PrimeScript RT master mix TaKaRa according to the instructions provided by the manufacturer. Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed.",
"Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed. The qRT-PCR primer sequences for NEAT1, NEAT1-2, IFN-, HTNV S segment, RIG-I, DDX60, -actin, and GAPDH were obtained from previous reports . . The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing.",
". The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing. HUVECs with a confluence of 80% in 6 wells were mock infected or infected with live or 60 Co-inactivated HTNV at an MOI of 1. RNAs were extracted as previously described at 24 hpi, and the quality was analyzed using FastQC software by the Beijing Genomics Institute BGI, Shenzhen, China . Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded.",
"Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded. Tag counts were normalized to TPM transcripts per million by dividing the raw tag count by the total number of tags from each library and multiplying by 1 million. To avoid the possible noise signal from high-throughput sequencing, the genes with average TPM of less than 1 in these three states were excluded. In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01.",
"In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01. Genes were selected as differentially expressed using a P value threshold of 0.01. FISH and immunofluorescence assays IFA . Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature.",
"Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature. Prehybridization was performed with lncRNA FISH probe mix at 37°C for 30 min, and then hybridization was performed by adding NEAT1-2 FISH probe mix and incubating the mixture at 37°C overnight. After washing with 4ϫ, 2ϫ, and 1ϫ SSC, the cell nuclei were stained with DAPI 4=,6-diamidino-2-phenylindole . Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently.",
"Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently. The cells were fixed with 4% PFA for 10 min and permeabilized with 0.1% Triton X-100 for 15 min. Primary Abs were added and incubated at 37°C for 2 h. After five washes with DPBS, secondary Cy3-or fluorescein isothiocyanate FITC -conjugated goat anti-rabbit or goat anti-mouse IgG Sangon, Shanghai, China was added and incubated at 37°C for 2 h. Cell nuclei were stained with DAPI. Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue .",
"Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue . The samples were then boiled at 95°C for 10 min. The lysates were resolved by 10%, 12%, or 15% SDS-PAGE and transferred to polyvinylidene fluoride PVDF membranes Millipore . The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA .",
"The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA . ICW assay. The In-Cell Western ICW assay was performed using an Odyssey imaging system Li-Cor according to the manufacturer's instructions. HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1.",
"HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1. At 48 hpi, HUVECs were washed twice with ice-cold DPBS, fixed with 4% PFA for 10 min, and permeabilized with 1.0% Triton X-100 for 15 min. Cells were added with Li-Cor Odyssey blocking solution at room temperature for 30 min and incubated at 4°C overnight with mouse IgG MAb 1A8 against HTNV NP together with rabbit IgG antibody against -actin, both of which were diluted in PBS containing 3% bovine serum albumin BSA; HyClone . Subsequently, the cells were washed and stained with goat anti-mouse IgG IRDye 800 antibody 1:5,000; Li-Cor and goat anti-rabbit"
] | 2,652 | 4,220 |
What plays a role in innate immunity to Hantavirus infection? | long noncoding RNAs (lncRNAs) | [
"Hantavirus infection, which causes zoonotic diseases with a high mortality rate in humans, has long been a global public health concern. Over the past decades, accumulating evidence suggests that long noncoding RNAs lncRNAs play key regulatory roles in innate immunity. However, the involvement of host lncRNAs in hantaviral control remains uncharacterized. In this study, we identified the lncRNA NEAT1 as a vital antiviral modulator. NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection.",
"NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection. Further investigation suggested that NEAT1 served as positive feedback for RIG-I signaling. HTNV infection activated NEAT1 transcription through the RIG-I–IRF7 pathway, whereas NEAT1 removed the transcriptional inhibitory effects of the splicing factor proline- and glutamine-rich protein SFPQ by relocating SFPQ to paraspeckles, thus promoting the expression of RIG-I and DDX60. RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections.",
"RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections. IMPORTANCE Hantaviruses have attracted worldwide attention as archetypal emerging pathogens. Recently, increasing evidence has highlighted long noncoding RNAs lncRNAs as key regulators of innate immunity; however, their roles in hantavirus infection remain unknown. In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling.",
"In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling. This lncRNA was induced by HTNV through the RIG-I–IRF7 pathway in a time- and dose-dependent manner and promoted HTNV-induced IFN production by facilitating RIG-I and DDX60 expression. Intriguingly, NEAT1 relocated SFPQ and formed paraspeckles after HTNV infection, which might reverse inhibitive effects of SFPQ on the transcription of RIG-I and DDX60. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections.",
"To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections. Text: glycoprotein GP , and viral RNA-dependent polymerase protein RdRp , respectively. Humans become infected by inhaling contaminated aerosols or by coming into contact with rodent excreta, and they develop two severe acute diseases, namely, hemorrhagic fever with renal syndrome HFRS and hantavirus pulmonary syndrome HPS . . Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . .",
". Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . . Chinese HFRS cases, mainly caused by Hantaan virus HTNV infection, account for approximately 90% of all global cases, with a mortality rate ranging from 0.1 to 15% . . Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions.",
"Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions. Cellular pathogen recognition receptors PRRs , including Toll-like receptors TLRs and RIG-I like receptors RLRs , can detect distinct pathogen-associated molecular patterns PAMPs and trigger the expression of IFNs and cytokines. RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression.",
"RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression. lncRNA nuclear paraspeckle assembly transcript 1 NEAT1 is an essential architectural constituent of paraspeckles in the mammalian nucleus, interacting with Drosophila DBHS RNA-binding proteins such as the splicing factor proline-and glutamine-rich protein SFPQ and the non-POU domain-containing, octamer-binding protein NONO/p54 . . To date, two isoform transcripts of the NEAT1 gene have been identified, namely, the 3.7-kb NEAT1-1 MEN and the 23-kb NEAT1-2 MEN Fig. 1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . .",
"1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . . , promoting cancer formation in mice by dampening oncogene-dependent activation of p53 . . Nevertheless, studies assessing the function of NEAT1 in viral infections are scarce. Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication.",
"Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication. Further investigation showed that NEAT1 promoted RIG-I and DDX60 expression by relocating SFPQ and removing the transcriptional inhibitory effects of SFPQ, which are critical for IFN responses against HTNV infection. We also found that RIG-I signaling, rather than TLR3 and TLR4, accounted for the elevation of HTNV-induced NEAT1. Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection.",
"Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection. As shown in Fig. 1B , the NEAT1 level in the HTNV group was higher than that in the mock group P ϭ 6.86 ϫ 10 Ϫ13 , false discovery rate FDR ϭ 9.75 ϫ 10 Ϫ12 or the 60 Co-inactivated HTNV group P ϭ 1.75 ϫ 10 Ϫ14 , FDR ϭ 3.10 ϫ 10 Ϫ13 ; however, the difference between the 60 Co-inactivated HTNV group and the mock group was not significant P ϭ 0.21034, FDR ϭ 0.58211 . To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig.",
"To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig. 1A , were applied to quantify NEAT1 RNA isoforms by quantitative real-time PCR qRT-PCR . It has been reported that NEAT1-2 rather than NEAT1-1 plays a key regulatory role in paraspeckle formation . , and we also found that elevated NEAT1 levels depend on live HTNV infection rather than 60 Co-inactivated HTNV stimulation Fig. 1C . Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D .",
"Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D . To further investigate whether NEAT1 expression was altered in other cell lines, HEK293, HeLa, and A549 cells were used. All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G .",
"All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G . Similar to the data obtained from HUVECs, NEAT1 was indeed upregulated by HTNV at a multiplicity of infection MOI of 1 beginning at 24 hpi in HUVECs and A549, HEK293, and HeLa cells, and the increasing tendency occurred in a time-dependent manner Fig. 1H . Of note, the NEAT1 elevation at 2 hpi might have been unrelated to the virus but resulted in cellular stress responses. Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I .",
"Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I . NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. The abovedescribed data showed that HTNV infection increased NEAT1, and we wondered how NEAT1 could reciprocally influence HTNV replication. The small interfering RNA siRNA transfection efficiency in HUVECs was confirmed by flow cytometry, and NEAT1 expression was significantly decreased, as assessed by qRT-PCR after RNA interference RNAi Fig. 2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2.",
"2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2. Compared with the cells transfected with control siRNA negative control NC , HUVECs with si-NEAT1 could dramatically promote HTNV NP production, and NP expression seemed to be related to the amount of applied si-NEAT1 Fig. 2B . Intriguingly, depletion of NEAT1-2 alone could mimic the antiviral effects of simultaneous NEAT1-1 and NEAT1-2 silencing Fig. 2C , indicating that NEAT1-2 was critical for the antiviral responses. Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing.",
"Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing. On the other hand, plasmids, of which pCMV-NEAT1-1 is transcribed into the 3.7-kb NEAT1-1 MEN and pCMV-NEAT1-2 is transcribed into the 2-to 3-kb NEAT1-2 MEN , were applied to directly investigate the role of NEAT1 in HTNV infection Fig. 2F . Surprisingly, we found NEAT1-1 overexpression restricted NEAT1-2 transcription Fig. 2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G .",
"2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G . Furthermore, overexpression of NEAT1-2 instead of NEAT1-1 could efficiently suppress HTNV replication Fig. 2H . NEAT1-1 upregulation even aggravated HTNV infection Fig. 2H , which may be the result of downregulation of NEAT1-2. Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig.",
"Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig. 2J were limited in HUVECs in which NEAT1-2 was ectopically expressed in comparison to those transfected with control vector or pCMV-NEAT1-1. These data further showed that NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. Alteration of NEAT1-2 affects HTNV-induced IFN expression in HUVECs. IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . .",
"IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . . It has been reported that the GnT of hantaviruses suppressed IFN- expression of host cells at an early stage of infection . . Here, we also found that HUVECs could not efficiently produce IFN- until 12 hpi at an MOI of 0.1 or until 24 hpi at an MOI of 1 Fig. 3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig.",
"3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig. 3B , suggesting that NEAT1-2 expression increased just before IFN production. We found that expression of endogenous IFN- mRNA was much lower in cells transfected with si-NEAT1-2 at MOIs of both 0.1 Fig. 3C and 1 Fig. 3D than in those transfected with control siRNA NC . In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E .",
"In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E . More importantly, HUVECs transfected with pCMV-NEAT1-2 conspicuously increased IFN- gene expression compared with those cells with vector plasmids at 12 hpi MOI ϭ 1 , demonstrating that NEAT1-2 overexpression accelerated robust IFN responses in host cells against HTNV infection. With a dual luciferase reporter system Twenty-four hours after transfection, the cells expressing FAM were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1.",
"Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the NC group . NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g .",
"NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 0.1 for 48 h. The expression of HTNV NP was measured by Western blotting. C HUVECs were treated as described for panel A, right, but at an MOI of 0.1. In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.",
"In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NC group . D HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group .",
"The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . E HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g .",
"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g . Twenty-four hours after transfection, the cells expressing green fluorescent protein GFP were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with control plasmids vector , pCMV-NEAT1-1, or pCMV-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig.",
"Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig. 3F . These results showed that NEAT1-2 regulated HTNV-induced IFN- expression. To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig.",
"To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig. 3G . In addition, in cells with high NEAT1-2 expression, treatment with neutralizing antibodies NAbs of IFN-␣ and IFN- could counteract the antiviral effects of NEAT1-2 MOI ϭ 1 , and the compensatory effects were dependent on the magnitude of the NAbs. Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production.",
"Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production. PRRs maintain a vital role in the promotion of IFN responses, and we conjectured that NEAT1 might amplify IFN responses by modulating these molecules. TLR3, TLR4, and RIG-I have been shown to recognize HTNV infection . . DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate .",
". DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate . , Here, we found that multiple Toll-like receptors like TLR1, TLR2, TLR3, and TLR4, as well as MDA5, were increased after HTNV infection, but none of them were influenced by silencing NEAT1-2 Fig. 4A . The upregulated RIG-I and DDX60 were blocked in the cells with low NEAT1-2 expression after HTNV infection Fig. 4A . HUVECs with declining NEAT1-2 expression showed gradually decreasing expression of RIG-I and DDX60 Fig. 4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C .",
"4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C . These data indicated that NEAT1-2 could positively modulate RIG-I and DDX60 expression, while the role of RIG-I and DDX60 upon HTNV infection is obscure. We then found that RIG-I and DDX60 colocalized after HTNV infection Fig. 4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown .",
"4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown . Simultaneously knocking down RIG-I and DDX60 significantly promoted HTNV NP expression Fig. 4E , and knockdown of both of them could greatly affect IFN- expression Fig. 4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig.",
"4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig. 4H , indicating that efficient anti-HTNV responses might depend on the interactive effects of DDX60 and RIG-I. More importantly, RIG-I or/and DDX60 overexpression enhanced HTNV-induced IFN- expression, and they had synergistic effects on IFN- production Fig. 4I and J . Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60.",
"Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60. NEAT1 was found to interact with SFPQ by RNA immunoprecipitation RIP after HTNV infection Fig. 5A , indicating that modulatory effects of NEAT1 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively .",
"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1 for 48 h. The expression of HTNV NP was measured by Western blotting. H HUVECs were treated as described for panel F, right. In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.",
"In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . H HUVECs were treated as described for panel F, right. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group .",
"The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . I HUVECs were treated as described for panel F, right. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ.",
"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ. Interestingly, the protein level of SFPQ, as well as another paraspeckle-forming constituent, NONO, remained unchanged after HTNV infection Fig. 5B or after NEAT1 overexpression and knockdown Fig. 5C . However, SFPQ became centralized rather than diffuse in the nucleus after HTNV infection Fig. 5D . The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig.",
"The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig. 5F and G , which might have been related to the increase in RIG-I Fig. 5H and DDX60 Fig. 5I . SFPQ has been suggested to bind to the promoter region of RIG-I and DDX60 ., thus preventing the expression of RIG-I and DDX60. Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription.",
"Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription. Interestingly, overexpression of the S or M segment of HTNV in HEK293 cells failed to induce NEAT1 expression, suggesting that NEAT1 transcription was closely related to live viral replication Fig. 6A . Of note, the upregulation of NEAT1 by HTNV could not be reversed by applying IFN-I neutralizing antibodies Fig. 6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig.",
"6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig. 6C, D, and E or cytokines Fig. 6F and G . We conjectured that NEAT1 expression was related to the activation of PRRs. By knocking down several PRRs, we found that the RIG-I and TLR4 pathways played important roles in HTNV-induced NEAT1 upregulation Fig. 7A . Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C .",
"Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C . Moreover, using STAT1 as a positive control, we found that the transcription factor IRF7, rather than IRF3 and p65, translocated into the nucleus in HTNV-infected HUVECs at 2 dpi Fig. 7D . Furthermore, IRF7 knockdown blocked HTNV-induced NEAT1 upregulation Fig. 7E . Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice.",
"Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice. Although cell-based experiments revealed that NEAT1-2 is a crucial regulator of innate antihantaviral responses, its function in vivo has remained unclear. To address this question, we intravenously injected siRNAs targeting mouse NEAT1-2 at 1 day before HTNV infection. NEAT1-2 expression levels in the liver, kidney, and spleen were reduced at 2 dpi Fig. 8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions.",
"8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions. Body weight loss in NEAT1-2-depleted mice was observed from 2 dpi to 5 dpi, and the IFN production in serum was remarkably decreased in the NEAT1-2 silenced group than those in the NC group at 3 dpi Fig. 8B . As expected, NEAT1-2 knockdown mice showed considerably higher HTNV NP levels in the liver, spleen, and kidney at 3 dpi Fig. 8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D .",
"8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D . In addition, reduced inflammatory cell filtration but increased tissue injury was found in NEAT1-2 knockdown mice during the early stage of infection Fig. 8E . Infiltration of macrophages in the spleen was attenuated Fig. 8F , and the activation of macrophages was also suppressed by flow cytometry; data not shown . Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G .",
"Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G . Nevertheless, NEAT1-2 silencing had no effect on the production of neutralizing antibodies at 7 dpi data not shown . The above-described findings indicated that NEAT1-2 depletion might influence multiple aspects of the innate immune response in HTNV-infected mice. Innate immunity is a phylogenetically ancient and conserved system that counteracts invading microbes, the regulatory mechanism of which is sophisticated and complex. Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . .",
"Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . . In this report, we first demonstrated that NEAT1 was induced by HTNV through the RIG-I-IRF7 pathway and served as positive feedback for RIG-I signaling. Using DGE analysis, we observed upregulated NEAT1 and confirmed its alteration in To further determine the function of NEAT1 after HTNV infection in vivo, mice were injected intravenously with si-NEAT1-2 1 g/g or nontarget control siRNA NC 1 g/g ; 1 day later, they were infected with HTNV 100 LD 50 by intramuscular injection. A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group .",
"A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group . B The effects of NEAT1 on HTNV virulence in mice were determined by body weight loss from 0 to 10 dpi left panel, n ϭ 10 in each group . The IFN- in sera of different groups was measured by ELISA at 3dpi right panel, n ϭ 8 in each group . Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi.",
"Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi. Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . D NEAT1 effects on HTNV infection kinetics at 3 dpi were determined by testing the HTNV titers in livers, spleens, and kidneys. Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group .",
"Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group . After HTNV infection, livers in the NC group showed inflammatory cell infiltration in certain regions, while those in the si-NEAT1-2 group showed slight acute viral hepatitis. Spleens in the NC group showed lymph node hyperplasia, while those in the si-NEAT1-2 group were severely congestive. Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented.",
"Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented. G CD3 ϩ CD8 ϩ IFN-␥ ϩ T cells were analyzed by flow cytometry at 3 dpi, and the results obtained for three mice in each group are presented. different cell lines. To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression.",
"To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression. By virtue of RNAi, the RIG-I-IRF7 pathway was confirmed to be necessary for HTNV-triggered NEAT1 elevation. Recently, large-scale transcriptomic studies identified numerous noncoding transcripts in the mammalian genome, which were speculated to influence diverse biological processes. Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression .",
"Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression . . TLR2 activation or tumor necrosis factor alpha TNF-␣ stimulation induces transcription of the lncRNA THRIL, the downregulation of which impairs TNF-␣ and IL-6 secretion . . TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . .",
". TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . . The lncRNA Lethe, triggered by TNF-␣ and IL-1, acts as a negative feedback regulator of NF-B signaling . . The roles of lncRNAs in host-virus interactions have been progressively unveiled. Various viruses, such as influenza virus IAV , coronavirus, enterovirus, human immunodeficiency virus HIV , hepatitis B virus HBV , hepatitis C virus HCV , Japanese encephalitis virus JEV , and rabies virus, have been reported to activate the transcription of different lncRNAs in host cells 11, . . . .",
". . . Importantly, multiple lncRNAs have been shown to affect the IFN response in recent years and have gradually become hot spots in the field of antiviral research. NeST was shown to enhance IFN-␥ production, controlling the susceptibility of mice to persistent Theiler's virus infection as well as resistance to Salmonella enterica serovar Typhimurium infection . . Both CMPK2 and NRAV were identified as negative regulators of IFN immune reactions. CMPK2, induced by IFN-␣ or HCV infection, suppresses various ISGs, the knockdown of which dramatically blocks HCV replication . . NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . .",
". NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . . Numerous lncRNAs, including lnc-ISG15 and ISR2, respond to IFNs such as ISGs, although their actual function requires further investigation . . Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection.",
"Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection. NEAT1 has been reported to interact with Drosophila DBHS RNA-binding proteins e.g., SFPQ, NONO-p54nrb, and PSPC1 , recruiting them to paraspeckles, a nuclear substructure found in all cultured and primary cells except embryonic stem cells . . The versatile function of NEAT1 is rapidly progressing in multiple areas of biology. NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . .",
"NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . . NEAT1 also participates in neurodegenerative diseases such as Huntington's disease . and seems to potentially contribute to the elevated production of a number of cytokines and chemokines in patients with systemic lupus erythematosus SLE . . Furthermore, poly I·C can activate NEAT1 transcription through the TLR3 pathway, whereas NEAT1 positively regulates IL-8 transcription and potentially affects the expression of multiple ISGs after poly I·C stimulation . . In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . .",
"In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . . However, the role of NEAT1 in hantaviral infection remains unclear. In this report, NEAT1 has been identified as an important regulator of the host innate immune system against HTNV infection. Elevated NEAT1 promotes IFN secretion, most likely by enhancing RIG-I and DDX60 expression. DDX60, a DEXD/H box RNA helicase similar to Saccharomyces cerevisiae Ski2, is induced after viral infection . . DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response.",
". DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response. However, the antiviral effects of DDX60 seem to vary among viruses . . We found that NEAT1-regulated DDX60 was involved in IFN production in response to HTNV infection. In HTNV-infected cells, double-stranded RNA dsRNA could not be detected, and it is unclear how host PRRs, especially RIG-I, recognize HTNV invasion . . Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation.",
". Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation. Of note, we applied multiple cell lines to explore the role of NEAT1 during HTNV infection. HTNV primarily targets vascular endothelial cells in vivo and contributes to the increased vascular permeability and coagulation disorders in HFRS; hence, HUVECs are the most common in vitro cell model to study host innate immunity against HTNV infection or viral pathogenesis . . EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies .",
". EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies . . A549 cells were once used to isolate HTNV, and they were confirmed to be a mature model of infection . . . . . Additionally, Huh 7.0 and Huh 7.5 RIG-I Ϫ cells used in our study have been reported to be infected by HTNV by Lee et al. . and can be used as a cell model to study immune responses against HTNV replication . . Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV.",
". Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV. Using qRT-PCR, Western blotting, and immunofluorescence assays, we have also shown that both HEK293 and HeLa cells can be infected by HTNV. To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production.",
"To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production. Alterations in the relative fluorescence intensity of NP after silencing or overexpressing NEAT1-2 did not seem to be as remarkable as qRT-PCR or Western blot analysis results. The NP spotted and exhibited in the ICW results forms obvious stains that mimic PFU. However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays.",
"However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays. The RNAi studies in vivo are encouraging Fig. 8 , but the NC used by our group was not mutated si-NEAT1-2 i.e., same sense strand, but with a point mutation in the targeting strand . The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling.",
"The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling. After observing that NEAT1 can regulate IFN expression by HTNV infection, we were interested in the function of NEAT1. We noticed that silencing NEAT1-2 or ectopically expressing NEAT1-2 could not inhibit or enhance IFN expression without HTNV infection Fig. 3F , which indicated that NEAT1-2 could not directly affect IFN- expression. This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression.",
"This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression. Recent findings have shown that RIG-I signaling is essential for an efficient polyfunctional T cell response during IAV infection . . Indeed, we found that the function of T cells was suppressed after NEAT1-2 depletion in our animal experiments Fig. 8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection.",
"8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection. HTNV infection induced NEAT1 expression through the RIG-I-IRF7 pathway, while NEAT1 displayed positive feedback for RIG-I signaling. NEAT1 relocated SFPQ from the potential promoter region of several antiviral genes to the paraspeckles, removing the transcriptional inhibitory effects of SFPQ. This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 .",
"This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 . nontarget control i.e., negative control NC or targeted RIG-I and DDX60 were designed by Gene-Pharma as follows: NC, 5=- UUCUUCGAACGUGUCACGUTT-3=; si-RIG-I-1, 5=-GCCCAUUUAAACCAAGAAATT-3=, si-RIG-I-2, 5=-GGUGGAGGAUAUUUGAACUTT-3=, and si-RIG-I-3, 5=-CCCAACGAUAUCAUUUCUTT-3=; si-DDX60-1, 5=-GUCCAGGUGUCAGUUUGAUTT-3=, si-DDX60-2, 5=-CCGAAGUGAAGAAGGUAAATT-3=, and si-DDX60-3, 5=-GAUGGAUGCUAGGAAAUAUTT-3=. The pCMV-NEAT1-1 and pCMV-NEAT1-2 plasmids, which transcribe NEAT1-1 and NEAT1-2, respectively, were provided by Nakagawa Shinichi . . The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . .",
". The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . . Abs against RIG-I, IRF3, IRF7, p65, and STAT1, as well as the neutralizing antibodies against IFN-␣ and IFN-, were purchased from Abcam Cambridge, MA, USA . Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China .",
"Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China . The Abs targeting CD11b, F4/80, CD3, CD8, IFN-␥, inducible nitric oxide synthase iNOS , and CD206 for flow cytometry were purchased from BD Biosciences San Jose, CA, USA . IFN-␣, -, and -␥, TNF-␣, and IL-1 were from PeperoTech Rocky Hill, NJ . ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis.",
"ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis. Total cellular RNAs were extracted with RNAiso TaKaRa, Dalian, China , the concentration of which was measured using a NanoDrop 1000 spectrophotometer. Reverse transcription RT was then performed with PrimeScript RT master mix TaKaRa according to the instructions provided by the manufacturer. Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed.",
"Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed. The qRT-PCR primer sequences for NEAT1, NEAT1-2, IFN-, HTNV S segment, RIG-I, DDX60, -actin, and GAPDH were obtained from previous reports . . The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing.",
". The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing. HUVECs with a confluence of 80% in 6 wells were mock infected or infected with live or 60 Co-inactivated HTNV at an MOI of 1. RNAs were extracted as previously described at 24 hpi, and the quality was analyzed using FastQC software by the Beijing Genomics Institute BGI, Shenzhen, China . Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded.",
"Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded. Tag counts were normalized to TPM transcripts per million by dividing the raw tag count by the total number of tags from each library and multiplying by 1 million. To avoid the possible noise signal from high-throughput sequencing, the genes with average TPM of less than 1 in these three states were excluded. In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01.",
"In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01. Genes were selected as differentially expressed using a P value threshold of 0.01. FISH and immunofluorescence assays IFA . Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature.",
"Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature. Prehybridization was performed with lncRNA FISH probe mix at 37°C for 30 min, and then hybridization was performed by adding NEAT1-2 FISH probe mix and incubating the mixture at 37°C overnight. After washing with 4ϫ, 2ϫ, and 1ϫ SSC, the cell nuclei were stained with DAPI 4=,6-diamidino-2-phenylindole . Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently.",
"Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently. The cells were fixed with 4% PFA for 10 min and permeabilized with 0.1% Triton X-100 for 15 min. Primary Abs were added and incubated at 37°C for 2 h. After five washes with DPBS, secondary Cy3-or fluorescein isothiocyanate FITC -conjugated goat anti-rabbit or goat anti-mouse IgG Sangon, Shanghai, China was added and incubated at 37°C for 2 h. Cell nuclei were stained with DAPI. Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue .",
"Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue . The samples were then boiled at 95°C for 10 min. The lysates were resolved by 10%, 12%, or 15% SDS-PAGE and transferred to polyvinylidene fluoride PVDF membranes Millipore . The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA .",
"The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA . ICW assay. The In-Cell Western ICW assay was performed using an Odyssey imaging system Li-Cor according to the manufacturer's instructions. HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1.",
"HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1. At 48 hpi, HUVECs were washed twice with ice-cold DPBS, fixed with 4% PFA for 10 min, and permeabilized with 1.0% Triton X-100 for 15 min. Cells were added with Li-Cor Odyssey blocking solution at room temperature for 30 min and incubated at 4°C overnight with mouse IgG MAb 1A8 against HTNV NP together with rabbit IgG antibody against -actin, both of which were diluted in PBS containing 3% bovine serum albumin BSA; HyClone . Subsequently, the cells were washed and stained with goat anti-mouse IgG IRDye 800 antibody 1:5,000; Li-Cor and goat anti-rabbit"
] | 2,652 | 5,311 |
What evidence suggests that RIG-I and DDX60 collaborate to exert antiviral effects? | colocalized after HTNV infection | [
"Hantavirus infection, which causes zoonotic diseases with a high mortality rate in humans, has long been a global public health concern. Over the past decades, accumulating evidence suggests that long noncoding RNAs lncRNAs play key regulatory roles in innate immunity. However, the involvement of host lncRNAs in hantaviral control remains uncharacterized. In this study, we identified the lncRNA NEAT1 as a vital antiviral modulator. NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection.",
"NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection. Further investigation suggested that NEAT1 served as positive feedback for RIG-I signaling. HTNV infection activated NEAT1 transcription through the RIG-I–IRF7 pathway, whereas NEAT1 removed the transcriptional inhibitory effects of the splicing factor proline- and glutamine-rich protein SFPQ by relocating SFPQ to paraspeckles, thus promoting the expression of RIG-I and DDX60. RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections.",
"RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections. IMPORTANCE Hantaviruses have attracted worldwide attention as archetypal emerging pathogens. Recently, increasing evidence has highlighted long noncoding RNAs lncRNAs as key regulators of innate immunity; however, their roles in hantavirus infection remain unknown. In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling.",
"In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling. This lncRNA was induced by HTNV through the RIG-I–IRF7 pathway in a time- and dose-dependent manner and promoted HTNV-induced IFN production by facilitating RIG-I and DDX60 expression. Intriguingly, NEAT1 relocated SFPQ and formed paraspeckles after HTNV infection, which might reverse inhibitive effects of SFPQ on the transcription of RIG-I and DDX60. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections.",
"To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections. Text: glycoprotein GP , and viral RNA-dependent polymerase protein RdRp , respectively. Humans become infected by inhaling contaminated aerosols or by coming into contact with rodent excreta, and they develop two severe acute diseases, namely, hemorrhagic fever with renal syndrome HFRS and hantavirus pulmonary syndrome HPS . . Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . .",
". Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . . Chinese HFRS cases, mainly caused by Hantaan virus HTNV infection, account for approximately 90% of all global cases, with a mortality rate ranging from 0.1 to 15% . . Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions.",
"Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions. Cellular pathogen recognition receptors PRRs , including Toll-like receptors TLRs and RIG-I like receptors RLRs , can detect distinct pathogen-associated molecular patterns PAMPs and trigger the expression of IFNs and cytokines. RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression.",
"RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression. lncRNA nuclear paraspeckle assembly transcript 1 NEAT1 is an essential architectural constituent of paraspeckles in the mammalian nucleus, interacting with Drosophila DBHS RNA-binding proteins such as the splicing factor proline-and glutamine-rich protein SFPQ and the non-POU domain-containing, octamer-binding protein NONO/p54 . . To date, two isoform transcripts of the NEAT1 gene have been identified, namely, the 3.7-kb NEAT1-1 MEN and the 23-kb NEAT1-2 MEN Fig. 1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . .",
"1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . . , promoting cancer formation in mice by dampening oncogene-dependent activation of p53 . . Nevertheless, studies assessing the function of NEAT1 in viral infections are scarce. Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication.",
"Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication. Further investigation showed that NEAT1 promoted RIG-I and DDX60 expression by relocating SFPQ and removing the transcriptional inhibitory effects of SFPQ, which are critical for IFN responses against HTNV infection. We also found that RIG-I signaling, rather than TLR3 and TLR4, accounted for the elevation of HTNV-induced NEAT1. Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection.",
"Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection. As shown in Fig. 1B , the NEAT1 level in the HTNV group was higher than that in the mock group P ϭ 6.86 ϫ 10 Ϫ13 , false discovery rate FDR ϭ 9.75 ϫ 10 Ϫ12 or the 60 Co-inactivated HTNV group P ϭ 1.75 ϫ 10 Ϫ14 , FDR ϭ 3.10 ϫ 10 Ϫ13 ; however, the difference between the 60 Co-inactivated HTNV group and the mock group was not significant P ϭ 0.21034, FDR ϭ 0.58211 . To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig.",
"To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig. 1A , were applied to quantify NEAT1 RNA isoforms by quantitative real-time PCR qRT-PCR . It has been reported that NEAT1-2 rather than NEAT1-1 plays a key regulatory role in paraspeckle formation . , and we also found that elevated NEAT1 levels depend on live HTNV infection rather than 60 Co-inactivated HTNV stimulation Fig. 1C . Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D .",
"Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D . To further investigate whether NEAT1 expression was altered in other cell lines, HEK293, HeLa, and A549 cells were used. All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G .",
"All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G . Similar to the data obtained from HUVECs, NEAT1 was indeed upregulated by HTNV at a multiplicity of infection MOI of 1 beginning at 24 hpi in HUVECs and A549, HEK293, and HeLa cells, and the increasing tendency occurred in a time-dependent manner Fig. 1H . Of note, the NEAT1 elevation at 2 hpi might have been unrelated to the virus but resulted in cellular stress responses. Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I .",
"Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I . NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. The abovedescribed data showed that HTNV infection increased NEAT1, and we wondered how NEAT1 could reciprocally influence HTNV replication. The small interfering RNA siRNA transfection efficiency in HUVECs was confirmed by flow cytometry, and NEAT1 expression was significantly decreased, as assessed by qRT-PCR after RNA interference RNAi Fig. 2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2.",
"2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2. Compared with the cells transfected with control siRNA negative control NC , HUVECs with si-NEAT1 could dramatically promote HTNV NP production, and NP expression seemed to be related to the amount of applied si-NEAT1 Fig. 2B . Intriguingly, depletion of NEAT1-2 alone could mimic the antiviral effects of simultaneous NEAT1-1 and NEAT1-2 silencing Fig. 2C , indicating that NEAT1-2 was critical for the antiviral responses. Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing.",
"Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing. On the other hand, plasmids, of which pCMV-NEAT1-1 is transcribed into the 3.7-kb NEAT1-1 MEN and pCMV-NEAT1-2 is transcribed into the 2-to 3-kb NEAT1-2 MEN , were applied to directly investigate the role of NEAT1 in HTNV infection Fig. 2F . Surprisingly, we found NEAT1-1 overexpression restricted NEAT1-2 transcription Fig. 2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G .",
"2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G . Furthermore, overexpression of NEAT1-2 instead of NEAT1-1 could efficiently suppress HTNV replication Fig. 2H . NEAT1-1 upregulation even aggravated HTNV infection Fig. 2H , which may be the result of downregulation of NEAT1-2. Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig.",
"Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig. 2J were limited in HUVECs in which NEAT1-2 was ectopically expressed in comparison to those transfected with control vector or pCMV-NEAT1-1. These data further showed that NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. Alteration of NEAT1-2 affects HTNV-induced IFN expression in HUVECs. IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . .",
"IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . . It has been reported that the GnT of hantaviruses suppressed IFN- expression of host cells at an early stage of infection . . Here, we also found that HUVECs could not efficiently produce IFN- until 12 hpi at an MOI of 0.1 or until 24 hpi at an MOI of 1 Fig. 3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig.",
"3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig. 3B , suggesting that NEAT1-2 expression increased just before IFN production. We found that expression of endogenous IFN- mRNA was much lower in cells transfected with si-NEAT1-2 at MOIs of both 0.1 Fig. 3C and 1 Fig. 3D than in those transfected with control siRNA NC . In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E .",
"In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E . More importantly, HUVECs transfected with pCMV-NEAT1-2 conspicuously increased IFN- gene expression compared with those cells with vector plasmids at 12 hpi MOI ϭ 1 , demonstrating that NEAT1-2 overexpression accelerated robust IFN responses in host cells against HTNV infection. With a dual luciferase reporter system Twenty-four hours after transfection, the cells expressing FAM were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1.",
"Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the NC group . NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g .",
"NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 0.1 for 48 h. The expression of HTNV NP was measured by Western blotting. C HUVECs were treated as described for panel A, right, but at an MOI of 0.1. In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.",
"In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NC group . D HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group .",
"The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . E HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g .",
"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g . Twenty-four hours after transfection, the cells expressing green fluorescent protein GFP were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with control plasmids vector , pCMV-NEAT1-1, or pCMV-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig.",
"Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig. 3F . These results showed that NEAT1-2 regulated HTNV-induced IFN- expression. To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig.",
"To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig. 3G . In addition, in cells with high NEAT1-2 expression, treatment with neutralizing antibodies NAbs of IFN-␣ and IFN- could counteract the antiviral effects of NEAT1-2 MOI ϭ 1 , and the compensatory effects were dependent on the magnitude of the NAbs. Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production.",
"Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production. PRRs maintain a vital role in the promotion of IFN responses, and we conjectured that NEAT1 might amplify IFN responses by modulating these molecules. TLR3, TLR4, and RIG-I have been shown to recognize HTNV infection . . DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate .",
". DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate . , Here, we found that multiple Toll-like receptors like TLR1, TLR2, TLR3, and TLR4, as well as MDA5, were increased after HTNV infection, but none of them were influenced by silencing NEAT1-2 Fig. 4A . The upregulated RIG-I and DDX60 were blocked in the cells with low NEAT1-2 expression after HTNV infection Fig. 4A . HUVECs with declining NEAT1-2 expression showed gradually decreasing expression of RIG-I and DDX60 Fig. 4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C .",
"4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C . These data indicated that NEAT1-2 could positively modulate RIG-I and DDX60 expression, while the role of RIG-I and DDX60 upon HTNV infection is obscure. We then found that RIG-I and DDX60 colocalized after HTNV infection Fig. 4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown .",
"4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown . Simultaneously knocking down RIG-I and DDX60 significantly promoted HTNV NP expression Fig. 4E , and knockdown of both of them could greatly affect IFN- expression Fig. 4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig.",
"4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig. 4H , indicating that efficient anti-HTNV responses might depend on the interactive effects of DDX60 and RIG-I. More importantly, RIG-I or/and DDX60 overexpression enhanced HTNV-induced IFN- expression, and they had synergistic effects on IFN- production Fig. 4I and J . Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60.",
"Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60. NEAT1 was found to interact with SFPQ by RNA immunoprecipitation RIP after HTNV infection Fig. 5A , indicating that modulatory effects of NEAT1 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively .",
"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1 for 48 h. The expression of HTNV NP was measured by Western blotting. H HUVECs were treated as described for panel F, right. In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.",
"In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . H HUVECs were treated as described for panel F, right. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group .",
"The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . I HUVECs were treated as described for panel F, right. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ.",
"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ. Interestingly, the protein level of SFPQ, as well as another paraspeckle-forming constituent, NONO, remained unchanged after HTNV infection Fig. 5B or after NEAT1 overexpression and knockdown Fig. 5C . However, SFPQ became centralized rather than diffuse in the nucleus after HTNV infection Fig. 5D . The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig.",
"The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig. 5F and G , which might have been related to the increase in RIG-I Fig. 5H and DDX60 Fig. 5I . SFPQ has been suggested to bind to the promoter region of RIG-I and DDX60 ., thus preventing the expression of RIG-I and DDX60. Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription.",
"Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription. Interestingly, overexpression of the S or M segment of HTNV in HEK293 cells failed to induce NEAT1 expression, suggesting that NEAT1 transcription was closely related to live viral replication Fig. 6A . Of note, the upregulation of NEAT1 by HTNV could not be reversed by applying IFN-I neutralizing antibodies Fig. 6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig.",
"6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig. 6C, D, and E or cytokines Fig. 6F and G . We conjectured that NEAT1 expression was related to the activation of PRRs. By knocking down several PRRs, we found that the RIG-I and TLR4 pathways played important roles in HTNV-induced NEAT1 upregulation Fig. 7A . Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C .",
"Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C . Moreover, using STAT1 as a positive control, we found that the transcription factor IRF7, rather than IRF3 and p65, translocated into the nucleus in HTNV-infected HUVECs at 2 dpi Fig. 7D . Furthermore, IRF7 knockdown blocked HTNV-induced NEAT1 upregulation Fig. 7E . Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice.",
"Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice. Although cell-based experiments revealed that NEAT1-2 is a crucial regulator of innate antihantaviral responses, its function in vivo has remained unclear. To address this question, we intravenously injected siRNAs targeting mouse NEAT1-2 at 1 day before HTNV infection. NEAT1-2 expression levels in the liver, kidney, and spleen were reduced at 2 dpi Fig. 8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions.",
"8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions. Body weight loss in NEAT1-2-depleted mice was observed from 2 dpi to 5 dpi, and the IFN production in serum was remarkably decreased in the NEAT1-2 silenced group than those in the NC group at 3 dpi Fig. 8B . As expected, NEAT1-2 knockdown mice showed considerably higher HTNV NP levels in the liver, spleen, and kidney at 3 dpi Fig. 8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D .",
"8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D . In addition, reduced inflammatory cell filtration but increased tissue injury was found in NEAT1-2 knockdown mice during the early stage of infection Fig. 8E . Infiltration of macrophages in the spleen was attenuated Fig. 8F , and the activation of macrophages was also suppressed by flow cytometry; data not shown . Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G .",
"Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G . Nevertheless, NEAT1-2 silencing had no effect on the production of neutralizing antibodies at 7 dpi data not shown . The above-described findings indicated that NEAT1-2 depletion might influence multiple aspects of the innate immune response in HTNV-infected mice. Innate immunity is a phylogenetically ancient and conserved system that counteracts invading microbes, the regulatory mechanism of which is sophisticated and complex. Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . .",
"Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . . In this report, we first demonstrated that NEAT1 was induced by HTNV through the RIG-I-IRF7 pathway and served as positive feedback for RIG-I signaling. Using DGE analysis, we observed upregulated NEAT1 and confirmed its alteration in To further determine the function of NEAT1 after HTNV infection in vivo, mice were injected intravenously with si-NEAT1-2 1 g/g or nontarget control siRNA NC 1 g/g ; 1 day later, they were infected with HTNV 100 LD 50 by intramuscular injection. A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group .",
"A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group . B The effects of NEAT1 on HTNV virulence in mice were determined by body weight loss from 0 to 10 dpi left panel, n ϭ 10 in each group . The IFN- in sera of different groups was measured by ELISA at 3dpi right panel, n ϭ 8 in each group . Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi.",
"Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi. Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . D NEAT1 effects on HTNV infection kinetics at 3 dpi were determined by testing the HTNV titers in livers, spleens, and kidneys. Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group .",
"Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group . After HTNV infection, livers in the NC group showed inflammatory cell infiltration in certain regions, while those in the si-NEAT1-2 group showed slight acute viral hepatitis. Spleens in the NC group showed lymph node hyperplasia, while those in the si-NEAT1-2 group were severely congestive. Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented.",
"Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented. G CD3 ϩ CD8 ϩ IFN-␥ ϩ T cells were analyzed by flow cytometry at 3 dpi, and the results obtained for three mice in each group are presented. different cell lines. To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression.",
"To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression. By virtue of RNAi, the RIG-I-IRF7 pathway was confirmed to be necessary for HTNV-triggered NEAT1 elevation. Recently, large-scale transcriptomic studies identified numerous noncoding transcripts in the mammalian genome, which were speculated to influence diverse biological processes. Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression .",
"Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression . . TLR2 activation or tumor necrosis factor alpha TNF-␣ stimulation induces transcription of the lncRNA THRIL, the downregulation of which impairs TNF-␣ and IL-6 secretion . . TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . .",
". TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . . The lncRNA Lethe, triggered by TNF-␣ and IL-1, acts as a negative feedback regulator of NF-B signaling . . The roles of lncRNAs in host-virus interactions have been progressively unveiled. Various viruses, such as influenza virus IAV , coronavirus, enterovirus, human immunodeficiency virus HIV , hepatitis B virus HBV , hepatitis C virus HCV , Japanese encephalitis virus JEV , and rabies virus, have been reported to activate the transcription of different lncRNAs in host cells 11, . . . .",
". . . Importantly, multiple lncRNAs have been shown to affect the IFN response in recent years and have gradually become hot spots in the field of antiviral research. NeST was shown to enhance IFN-␥ production, controlling the susceptibility of mice to persistent Theiler's virus infection as well as resistance to Salmonella enterica serovar Typhimurium infection . . Both CMPK2 and NRAV were identified as negative regulators of IFN immune reactions. CMPK2, induced by IFN-␣ or HCV infection, suppresses various ISGs, the knockdown of which dramatically blocks HCV replication . . NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . .",
". NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . . Numerous lncRNAs, including lnc-ISG15 and ISR2, respond to IFNs such as ISGs, although their actual function requires further investigation . . Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection.",
"Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection. NEAT1 has been reported to interact with Drosophila DBHS RNA-binding proteins e.g., SFPQ, NONO-p54nrb, and PSPC1 , recruiting them to paraspeckles, a nuclear substructure found in all cultured and primary cells except embryonic stem cells . . The versatile function of NEAT1 is rapidly progressing in multiple areas of biology. NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . .",
"NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . . NEAT1 also participates in neurodegenerative diseases such as Huntington's disease . and seems to potentially contribute to the elevated production of a number of cytokines and chemokines in patients with systemic lupus erythematosus SLE . . Furthermore, poly I·C can activate NEAT1 transcription through the TLR3 pathway, whereas NEAT1 positively regulates IL-8 transcription and potentially affects the expression of multiple ISGs after poly I·C stimulation . . In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . .",
"In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . . However, the role of NEAT1 in hantaviral infection remains unclear. In this report, NEAT1 has been identified as an important regulator of the host innate immune system against HTNV infection. Elevated NEAT1 promotes IFN secretion, most likely by enhancing RIG-I and DDX60 expression. DDX60, a DEXD/H box RNA helicase similar to Saccharomyces cerevisiae Ski2, is induced after viral infection . . DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response.",
". DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response. However, the antiviral effects of DDX60 seem to vary among viruses . . We found that NEAT1-regulated DDX60 was involved in IFN production in response to HTNV infection. In HTNV-infected cells, double-stranded RNA dsRNA could not be detected, and it is unclear how host PRRs, especially RIG-I, recognize HTNV invasion . . Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation.",
". Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation. Of note, we applied multiple cell lines to explore the role of NEAT1 during HTNV infection. HTNV primarily targets vascular endothelial cells in vivo and contributes to the increased vascular permeability and coagulation disorders in HFRS; hence, HUVECs are the most common in vitro cell model to study host innate immunity against HTNV infection or viral pathogenesis . . EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies .",
". EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies . . A549 cells were once used to isolate HTNV, and they were confirmed to be a mature model of infection . . . . . Additionally, Huh 7.0 and Huh 7.5 RIG-I Ϫ cells used in our study have been reported to be infected by HTNV by Lee et al. . and can be used as a cell model to study immune responses against HTNV replication . . Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV.",
". Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV. Using qRT-PCR, Western blotting, and immunofluorescence assays, we have also shown that both HEK293 and HeLa cells can be infected by HTNV. To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production.",
"To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production. Alterations in the relative fluorescence intensity of NP after silencing or overexpressing NEAT1-2 did not seem to be as remarkable as qRT-PCR or Western blot analysis results. The NP spotted and exhibited in the ICW results forms obvious stains that mimic PFU. However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays.",
"However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays. The RNAi studies in vivo are encouraging Fig. 8 , but the NC used by our group was not mutated si-NEAT1-2 i.e., same sense strand, but with a point mutation in the targeting strand . The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling.",
"The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling. After observing that NEAT1 can regulate IFN expression by HTNV infection, we were interested in the function of NEAT1. We noticed that silencing NEAT1-2 or ectopically expressing NEAT1-2 could not inhibit or enhance IFN expression without HTNV infection Fig. 3F , which indicated that NEAT1-2 could not directly affect IFN- expression. This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression.",
"This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression. Recent findings have shown that RIG-I signaling is essential for an efficient polyfunctional T cell response during IAV infection . . Indeed, we found that the function of T cells was suppressed after NEAT1-2 depletion in our animal experiments Fig. 8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection.",
"8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection. HTNV infection induced NEAT1 expression through the RIG-I-IRF7 pathway, while NEAT1 displayed positive feedback for RIG-I signaling. NEAT1 relocated SFPQ from the potential promoter region of several antiviral genes to the paraspeckles, removing the transcriptional inhibitory effects of SFPQ. This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 .",
"This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 . nontarget control i.e., negative control NC or targeted RIG-I and DDX60 were designed by Gene-Pharma as follows: NC, 5=- UUCUUCGAACGUGUCACGUTT-3=; si-RIG-I-1, 5=-GCCCAUUUAAACCAAGAAATT-3=, si-RIG-I-2, 5=-GGUGGAGGAUAUUUGAACUTT-3=, and si-RIG-I-3, 5=-CCCAACGAUAUCAUUUCUTT-3=; si-DDX60-1, 5=-GUCCAGGUGUCAGUUUGAUTT-3=, si-DDX60-2, 5=-CCGAAGUGAAGAAGGUAAATT-3=, and si-DDX60-3, 5=-GAUGGAUGCUAGGAAAUAUTT-3=. The pCMV-NEAT1-1 and pCMV-NEAT1-2 plasmids, which transcribe NEAT1-1 and NEAT1-2, respectively, were provided by Nakagawa Shinichi . . The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . .",
". The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . . Abs against RIG-I, IRF3, IRF7, p65, and STAT1, as well as the neutralizing antibodies against IFN-␣ and IFN-, were purchased from Abcam Cambridge, MA, USA . Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China .",
"Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China . The Abs targeting CD11b, F4/80, CD3, CD8, IFN-␥, inducible nitric oxide synthase iNOS , and CD206 for flow cytometry were purchased from BD Biosciences San Jose, CA, USA . IFN-␣, -, and -␥, TNF-␣, and IL-1 were from PeperoTech Rocky Hill, NJ . ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis.",
"ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis. Total cellular RNAs were extracted with RNAiso TaKaRa, Dalian, China , the concentration of which was measured using a NanoDrop 1000 spectrophotometer. Reverse transcription RT was then performed with PrimeScript RT master mix TaKaRa according to the instructions provided by the manufacturer. Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed.",
"Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed. The qRT-PCR primer sequences for NEAT1, NEAT1-2, IFN-, HTNV S segment, RIG-I, DDX60, -actin, and GAPDH were obtained from previous reports . . The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing.",
". The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing. HUVECs with a confluence of 80% in 6 wells were mock infected or infected with live or 60 Co-inactivated HTNV at an MOI of 1. RNAs were extracted as previously described at 24 hpi, and the quality was analyzed using FastQC software by the Beijing Genomics Institute BGI, Shenzhen, China . Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded.",
"Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded. Tag counts were normalized to TPM transcripts per million by dividing the raw tag count by the total number of tags from each library and multiplying by 1 million. To avoid the possible noise signal from high-throughput sequencing, the genes with average TPM of less than 1 in these three states were excluded. In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01.",
"In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01. Genes were selected as differentially expressed using a P value threshold of 0.01. FISH and immunofluorescence assays IFA . Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature.",
"Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature. Prehybridization was performed with lncRNA FISH probe mix at 37°C for 30 min, and then hybridization was performed by adding NEAT1-2 FISH probe mix and incubating the mixture at 37°C overnight. After washing with 4ϫ, 2ϫ, and 1ϫ SSC, the cell nuclei were stained with DAPI 4=,6-diamidino-2-phenylindole . Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently.",
"Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently. The cells were fixed with 4% PFA for 10 min and permeabilized with 0.1% Triton X-100 for 15 min. Primary Abs were added and incubated at 37°C for 2 h. After five washes with DPBS, secondary Cy3-or fluorescein isothiocyanate FITC -conjugated goat anti-rabbit or goat anti-mouse IgG Sangon, Shanghai, China was added and incubated at 37°C for 2 h. Cell nuclei were stained with DAPI. Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue .",
"Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue . The samples were then boiled at 95°C for 10 min. The lysates were resolved by 10%, 12%, or 15% SDS-PAGE and transferred to polyvinylidene fluoride PVDF membranes Millipore . The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA .",
"The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA . ICW assay. The In-Cell Western ICW assay was performed using an Odyssey imaging system Li-Cor according to the manufacturer's instructions. HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1.",
"HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1. At 48 hpi, HUVECs were washed twice with ice-cold DPBS, fixed with 4% PFA for 10 min, and permeabilized with 1.0% Triton X-100 for 15 min. Cells were added with Li-Cor Odyssey blocking solution at room temperature for 30 min and incubated at 4°C overnight with mouse IgG MAb 1A8 against HTNV NP together with rabbit IgG antibody against -actin, both of which were diluted in PBS containing 3% bovine serum albumin BSA; HyClone . Subsequently, the cells were washed and stained with goat anti-mouse IgG IRDye 800 antibody 1:5,000; Li-Cor and goat anti-rabbit"
] | 2,652 | 5,312 |
What was the major contribution of this study? | the first study to describe the role of NEAT1 in HTNV infection | [
"Hantavirus infection, which causes zoonotic diseases with a high mortality rate in humans, has long been a global public health concern. Over the past decades, accumulating evidence suggests that long noncoding RNAs lncRNAs play key regulatory roles in innate immunity. However, the involvement of host lncRNAs in hantaviral control remains uncharacterized. In this study, we identified the lncRNA NEAT1 as a vital antiviral modulator. NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection.",
"NEAT1 was dramatically upregulated after Hantaan virus HTNV infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon IFN-β production and suppressed HTNV infection. Further investigation suggested that NEAT1 served as positive feedback for RIG-I signaling. HTNV infection activated NEAT1 transcription through the RIG-I–IRF7 pathway, whereas NEAT1 removed the transcriptional inhibitory effects of the splicing factor proline- and glutamine-rich protein SFPQ by relocating SFPQ to paraspeckles, thus promoting the expression of RIG-I and DDX60. RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections.",
"RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections. IMPORTANCE Hantaviruses have attracted worldwide attention as archetypal emerging pathogens. Recently, increasing evidence has highlighted long noncoding RNAs lncRNAs as key regulators of innate immunity; however, their roles in hantavirus infection remain unknown. In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling.",
"In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon IFN responses by acting as positive feedback for RIG-I signaling. This lncRNA was induced by HTNV through the RIG-I–IRF7 pathway in a time- and dose-dependent manner and promoted HTNV-induced IFN production by facilitating RIG-I and DDX60 expression. Intriguingly, NEAT1 relocated SFPQ and formed paraspeckles after HTNV infection, which might reverse inhibitive effects of SFPQ on the transcription of RIG-I and DDX60. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections.",
"To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections. Text: glycoprotein GP , and viral RNA-dependent polymerase protein RdRp , respectively. Humans become infected by inhaling contaminated aerosols or by coming into contact with rodent excreta, and they develop two severe acute diseases, namely, hemorrhagic fever with renal syndrome HFRS and hantavirus pulmonary syndrome HPS . . Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . .",
". Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China . . Chinese HFRS cases, mainly caused by Hantaan virus HTNV infection, account for approximately 90% of all global cases, with a mortality rate ranging from 0.1 to 15% . . Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions.",
"Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon IFN responses and immunocyte activation, provides the initial defense against viral invasions. Cellular pathogen recognition receptors PRRs , including Toll-like receptors TLRs and RIG-I like receptors RLRs , can detect distinct pathogen-associated molecular patterns PAMPs and trigger the expression of IFNs and cytokines. RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression.",
"RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear . . Long noncoding RNAs lncRNAs have emerged as important modulators of gene expression. lncRNA nuclear paraspeckle assembly transcript 1 NEAT1 is an essential architectural constituent of paraspeckles in the mammalian nucleus, interacting with Drosophila DBHS RNA-binding proteins such as the splicing factor proline-and glutamine-rich protein SFPQ and the non-POU domain-containing, octamer-binding protein NONO/p54 . . To date, two isoform transcripts of the NEAT1 gene have been identified, namely, the 3.7-kb NEAT1-1 MEN and the 23-kb NEAT1-2 MEN Fig. 1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . .",
"1A . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression . . . , promoting cancer formation in mice by dampening oncogene-dependent activation of p53 . . Nevertheless, studies assessing the function of NEAT1 in viral infections are scarce. Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication.",
"Here, the human umbilical vein endothelial cell HUVEC transcriptome was analyzed after HTNV infection by digital gene expression DGE profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon IFN- production and inhibited HTNV replication. Further investigation showed that NEAT1 promoted RIG-I and DDX60 expression by relocating SFPQ and removing the transcriptional inhibitory effects of SFPQ, which are critical for IFN responses against HTNV infection. We also found that RIG-I signaling, rather than TLR3 and TLR4, accounted for the elevation of HTNV-induced NEAT1. Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection.",
"Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection. As shown in Fig. 1B , the NEAT1 level in the HTNV group was higher than that in the mock group P ϭ 6.86 ϫ 10 Ϫ13 , false discovery rate FDR ϭ 9.75 ϫ 10 Ϫ12 or the 60 Co-inactivated HTNV group P ϭ 1.75 ϫ 10 Ϫ14 , FDR ϭ 3.10 ϫ 10 Ϫ13 ; however, the difference between the 60 Co-inactivated HTNV group and the mock group was not significant P ϭ 0.21034, FDR ϭ 0.58211 . To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig.",
"To confirm the profiling results, two primer pairs from the published literature . , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 Fig. 1A , were applied to quantify NEAT1 RNA isoforms by quantitative real-time PCR qRT-PCR . It has been reported that NEAT1-2 rather than NEAT1-1 plays a key regulatory role in paraspeckle formation . , and we also found that elevated NEAT1 levels depend on live HTNV infection rather than 60 Co-inactivated HTNV stimulation Fig. 1C . Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D .",
"Fluorescence in situ hybridization FISH with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection hpi Fig. 1D . To further investigate whether NEAT1 expression was altered in other cell lines, HEK293, HeLa, and A549 cells were used. All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G .",
"All these cells could be infected by HTNV Fig. 1E and F and generated hantavirus progeny Fig. 1G . Similar to the data obtained from HUVECs, NEAT1 was indeed upregulated by HTNV at a multiplicity of infection MOI of 1 beginning at 24 hpi in HUVECs and A549, HEK293, and HeLa cells, and the increasing tendency occurred in a time-dependent manner Fig. 1H . Of note, the NEAT1 elevation at 2 hpi might have been unrelated to the virus but resulted in cellular stress responses. Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I .",
"Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner Fig. 1I . NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. The abovedescribed data showed that HTNV infection increased NEAT1, and we wondered how NEAT1 could reciprocally influence HTNV replication. The small interfering RNA siRNA transfection efficiency in HUVECs was confirmed by flow cytometry, and NEAT1 expression was significantly decreased, as assessed by qRT-PCR after RNA interference RNAi Fig. 2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2.",
"2A . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 st-NEAT1-2 is specific for NEAT1-2. Compared with the cells transfected with control siRNA negative control NC , HUVECs with si-NEAT1 could dramatically promote HTNV NP production, and NP expression seemed to be related to the amount of applied si-NEAT1 Fig. 2B . Intriguingly, depletion of NEAT1-2 alone could mimic the antiviral effects of simultaneous NEAT1-1 and NEAT1-2 silencing Fig. 2C , indicating that NEAT1-2 was critical for the antiviral responses. Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing.",
"Consistent with those data, the expressions of HTNV mRNA of S segment Fig. 2D and HTNV titers Fig. 2E were increased after NEAT1 silencing. On the other hand, plasmids, of which pCMV-NEAT1-1 is transcribed into the 3.7-kb NEAT1-1 MEN and pCMV-NEAT1-2 is transcribed into the 2-to 3-kb NEAT1-2 MEN , were applied to directly investigate the role of NEAT1 in HTNV infection Fig. 2F . Surprisingly, we found NEAT1-1 overexpression restricted NEAT1-2 transcription Fig. 2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G .",
"2F . Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids Fig. 2G . Furthermore, overexpression of NEAT1-2 instead of NEAT1-1 could efficiently suppress HTNV replication Fig. 2H . NEAT1-1 upregulation even aggravated HTNV infection Fig. 2H , which may be the result of downregulation of NEAT1-2. Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig.",
"Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose TCID 50 test by ELISA, we found that expression of HTNV-specific mRNA Fig. 2I and HTNV titers Fig. 2J were limited in HUVECs in which NEAT1-2 was ectopically expressed in comparison to those transfected with control vector or pCMV-NEAT1-1. These data further showed that NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. Alteration of NEAT1-2 affects HTNV-induced IFN expression in HUVECs. IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . .",
"IFN- production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN- treatment after 24 hpi exerts little antiviral effect . . It has been reported that the GnT of hantaviruses suppressed IFN- expression of host cells at an early stage of infection . . Here, we also found that HUVECs could not efficiently produce IFN- until 12 hpi at an MOI of 0.1 or until 24 hpi at an MOI of 1 Fig. 3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig.",
"3A , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 Fig. 3B , suggesting that NEAT1-2 expression increased just before IFN production. We found that expression of endogenous IFN- mRNA was much lower in cells transfected with si-NEAT1-2 at MOIs of both 0.1 Fig. 3C and 1 Fig. 3D than in those transfected with control siRNA NC . In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E .",
"In contrast, overexpression of NEAT1 in HUVECs increased IFN- expression after HTNV infection MOI ϭ 1 at 24 and 48 hpi Fig. 3E . More importantly, HUVECs transfected with pCMV-NEAT1-2 conspicuously increased IFN- gene expression compared with those cells with vector plasmids at 12 hpi MOI ϭ 1 , demonstrating that NEAT1-2 overexpression accelerated robust IFN responses in host cells against HTNV infection. With a dual luciferase reporter system Twenty-four hours after transfection, the cells expressing FAM were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1.",
"Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 st-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the NC group . NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g .",
"NS, nonsignificant. B HUVECs in six-well plates were transfected with NC sequences the amount of Si-NEAT1-2 is considered 0 g or increasing amounts of si-NEAT1 0.1, 0.5, 1, and 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 0.1 for 48 h. The expression of HTNV NP was measured by Western blotting. C HUVECs were treated as described for panel A, right, but at an MOI of 0.1. In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.",
"In-cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NC group . D HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group .",
"The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . E HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g .",
"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group . F Left, HUVECs in six-well plates were transfected with vectors or pGFP 3 g . Twenty-four hours after transfection, the cells expressing green fluorescent protein GFP were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with control plasmids vector , pCMV-NEAT1-1, or pCMV-NEAT1-2 3 g . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig.",
"Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At Continued on next page NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN- promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN- gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN- expression Fig. 3F . These results showed that NEAT1-2 regulated HTNV-induced IFN- expression. To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig.",
"To explore whether the antihantavirus effects of NEAT1 were caused by IFN- alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN- at 12 hpi could efficiently block HTNV NP production MOI ϭ 0.1 , and such phenomena were also determined by the amount of applied IFN- Fig. 3G . In addition, in cells with high NEAT1-2 expression, treatment with neutralizing antibodies NAbs of IFN-␣ and IFN- could counteract the antiviral effects of NEAT1-2 MOI ϭ 1 , and the compensatory effects were dependent on the magnitude of the NAbs. Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production.",
"Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN- signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN- production. PRRs maintain a vital role in the promotion of IFN responses, and we conjectured that NEAT1 might amplify IFN responses by modulating these molecules. TLR3, TLR4, and RIG-I have been shown to recognize HTNV infection . . DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate .",
". DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate . , Here, we found that multiple Toll-like receptors like TLR1, TLR2, TLR3, and TLR4, as well as MDA5, were increased after HTNV infection, but none of them were influenced by silencing NEAT1-2 Fig. 4A . The upregulated RIG-I and DDX60 were blocked in the cells with low NEAT1-2 expression after HTNV infection Fig. 4A . HUVECs with declining NEAT1-2 expression showed gradually decreasing expression of RIG-I and DDX60 Fig. 4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C .",
"4B , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly Fig. 4C . These data indicated that NEAT1-2 could positively modulate RIG-I and DDX60 expression, while the role of RIG-I and DDX60 upon HTNV infection is obscure. We then found that RIG-I and DDX60 colocalized after HTNV infection Fig. 4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown .",
"4D , implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs data not shown . Simultaneously knocking down RIG-I and DDX60 significantly promoted HTNV NP expression Fig. 4E , and knockdown of both of them could greatly affect IFN- expression Fig. 4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig.",
"4F and G . Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects Fig. 4H , indicating that efficient anti-HTNV responses might depend on the interactive effects of DDX60 and RIG-I. More importantly, RIG-I or/and DDX60 overexpression enhanced HTNV-induced IFN- expression, and they had synergistic effects on IFN- production Fig. 4I and J . Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60.",
"Consequently, NEAT1 might regulate IFN- production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60. NEAT1 was found to interact with SFPQ by RNA immunoprecipitation RIP after HTNV infection Fig. 5A , indicating that modulatory effects of NEAT1 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively .",
"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group . G HUVECs in six-well plates were transfected with control plasmids vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively . Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1 for 48 h. The expression of HTNV NP was measured by Western blotting. H HUVECs were treated as described for panel F, right. In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test.",
"In-Cell Western ICW analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence NP/-actin was analyzed using Student's t test. n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . H HUVECs were treated as described for panel F, right. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group .",
"The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group . I HUVECs were treated as described for panel F, right. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ.",
"Values are means Ϯ SD n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group . might be involved in SFPQ. Interestingly, the protein level of SFPQ, as well as another paraspeckle-forming constituent, NONO, remained unchanged after HTNV infection Fig. 5B or after NEAT1 overexpression and knockdown Fig. 5C . However, SFPQ became centralized rather than diffuse in the nucleus after HTNV infection Fig. 5D . The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig.",
"The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus Fig. 5E and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication Fig. 5F and G , which might have been related to the increase in RIG-I Fig. 5H and DDX60 Fig. 5I . SFPQ has been suggested to bind to the promoter region of RIG-I and DDX60 ., thus preventing the expression of RIG-I and DDX60. Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription.",
"Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription. Interestingly, overexpression of the S or M segment of HTNV in HEK293 cells failed to induce NEAT1 expression, suggesting that NEAT1 transcription was closely related to live viral replication Fig. 6A . Of note, the upregulation of NEAT1 by HTNV could not be reversed by applying IFN-I neutralizing antibodies Fig. 6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig.",
"6B . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs Fig. 6C, D, and E or cytokines Fig. 6F and G . We conjectured that NEAT1 expression was related to the activation of PRRs. By knocking down several PRRs, we found that the RIG-I and TLR4 pathways played important roles in HTNV-induced NEAT1 upregulation Fig. 7A . Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C .",
"Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV Fig. 7B , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection Fig. 7C . Moreover, using STAT1 as a positive control, we found that the transcription factor IRF7, rather than IRF3 and p65, translocated into the nucleus in HTNV-infected HUVECs at 2 dpi Fig. 7D . Furthermore, IRF7 knockdown blocked HTNV-induced NEAT1 upregulation Fig. 7E . Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice.",
"Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice. Although cell-based experiments revealed that NEAT1-2 is a crucial regulator of innate antihantaviral responses, its function in vivo has remained unclear. To address this question, we intravenously injected siRNAs targeting mouse NEAT1-2 at 1 day before HTNV infection. NEAT1-2 expression levels in the liver, kidney, and spleen were reduced at 2 dpi Fig. 8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions.",
"8A . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions. Body weight loss in NEAT1-2-depleted mice was observed from 2 dpi to 5 dpi, and the IFN production in serum was remarkably decreased in the NEAT1-2 silenced group than those in the NC group at 3 dpi Fig. 8B . As expected, NEAT1-2 knockdown mice showed considerably higher HTNV NP levels in the liver, spleen, and kidney at 3 dpi Fig. 8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D .",
"8C . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group Fig. 8D . In addition, reduced inflammatory cell filtration but increased tissue injury was found in NEAT1-2 knockdown mice during the early stage of infection Fig. 8E . Infiltration of macrophages in the spleen was attenuated Fig. 8F , and the activation of macrophages was also suppressed by flow cytometry; data not shown . Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G .",
"Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi Fig. 8G . Nevertheless, NEAT1-2 silencing had no effect on the production of neutralizing antibodies at 7 dpi data not shown . The above-described findings indicated that NEAT1-2 depletion might influence multiple aspects of the innate immune response in HTNV-infected mice. Innate immunity is a phylogenetically ancient and conserved system that counteracts invading microbes, the regulatory mechanism of which is sophisticated and complex. Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . .",
"Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity . . In this report, we first demonstrated that NEAT1 was induced by HTNV through the RIG-I-IRF7 pathway and served as positive feedback for RIG-I signaling. Using DGE analysis, we observed upregulated NEAT1 and confirmed its alteration in To further determine the function of NEAT1 after HTNV infection in vivo, mice were injected intravenously with si-NEAT1-2 1 g/g or nontarget control siRNA NC 1 g/g ; 1 day later, they were infected with HTNV 100 LD 50 by intramuscular injection. A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group .",
"A To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. A The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi n ϭ 6 in each group . B The effects of NEAT1 on HTNV virulence in mice were determined by body weight loss from 0 to 10 dpi left panel, n ϭ 10 in each group . The IFN- in sera of different groups was measured by ELISA at 3dpi right panel, n ϭ 8 in each group . Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi.",
"Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . C Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers upper panels, n ϭ 8 in each group and qRT-PCR to assess HTNV S segment levels bottom panels, n ϭ 8 in each group at 3 dpi. Values are means Ϯ SD *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . D NEAT1 effects on HTNV infection kinetics at 3 dpi were determined by testing the HTNV titers in livers, spleens, and kidneys. Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group .",
"Values are means Ϯ SD n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group . E Hematoxylin and eosin H&E staining for mouse liver, spleen, or kidney specimens was performed 3 dpi, n ϭ 8 in each group . After HTNV infection, livers in the NC group showed inflammatory cell infiltration in certain regions, while those in the si-NEAT1-2 group showed slight acute viral hepatitis. Spleens in the NC group showed lymph node hyperplasia, while those in the si-NEAT1-2 group were severely congestive. Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented.",
"Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. F Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented. G CD3 ϩ CD8 ϩ IFN-␥ ϩ T cells were analyzed by flow cytometry at 3 dpi, and the results obtained for three mice in each group are presented. different cell lines. To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression.",
"To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression. By virtue of RNAi, the RIG-I-IRF7 pathway was confirmed to be necessary for HTNV-triggered NEAT1 elevation. Recently, large-scale transcriptomic studies identified numerous noncoding transcripts in the mammalian genome, which were speculated to influence diverse biological processes. Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression .",
"Among these noncoding RNAs ncRNAs , long noncoding RNAs lncRNAs emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 IL-6 secretion or represses ISG15 and CCL5 expression . . TLR2 activation or tumor necrosis factor alpha TNF-␣ stimulation induces transcription of the lncRNA THRIL, the downregulation of which impairs TNF-␣ and IL-6 secretion . . TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . .",
". TLR4 signaling in response to lipopolysaccharide LPS induces lncRNA IL-1-eRNA and IL-1-RBT46, the knockdown of which attenuates IL-1 and CXCL8 release . . The lncRNA Lethe, triggered by TNF-␣ and IL-1, acts as a negative feedback regulator of NF-B signaling . . The roles of lncRNAs in host-virus interactions have been progressively unveiled. Various viruses, such as influenza virus IAV , coronavirus, enterovirus, human immunodeficiency virus HIV , hepatitis B virus HBV , hepatitis C virus HCV , Japanese encephalitis virus JEV , and rabies virus, have been reported to activate the transcription of different lncRNAs in host cells 11, . . . .",
". . . Importantly, multiple lncRNAs have been shown to affect the IFN response in recent years and have gradually become hot spots in the field of antiviral research. NeST was shown to enhance IFN-␥ production, controlling the susceptibility of mice to persistent Theiler's virus infection as well as resistance to Salmonella enterica serovar Typhimurium infection . . Both CMPK2 and NRAV were identified as negative regulators of IFN immune reactions. CMPK2, induced by IFN-␣ or HCV infection, suppresses various ISGs, the knockdown of which dramatically blocks HCV replication . . NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . .",
". NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo . . Numerous lncRNAs, including lnc-ISG15 and ISR2, respond to IFNs such as ISGs, although their actual function requires further investigation . . Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection.",
"Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection. NEAT1 has been reported to interact with Drosophila DBHS RNA-binding proteins e.g., SFPQ, NONO-p54nrb, and PSPC1 , recruiting them to paraspeckles, a nuclear substructure found in all cultured and primary cells except embryonic stem cells . . The versatile function of NEAT1 is rapidly progressing in multiple areas of biology. NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . .",
"NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer . . . . NEAT1 also participates in neurodegenerative diseases such as Huntington's disease . and seems to potentially contribute to the elevated production of a number of cytokines and chemokines in patients with systemic lupus erythematosus SLE . . Furthermore, poly I·C can activate NEAT1 transcription through the TLR3 pathway, whereas NEAT1 positively regulates IL-8 transcription and potentially affects the expression of multiple ISGs after poly I·C stimulation . . In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . .",
"In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element INS containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication . . However, the role of NEAT1 in hantaviral infection remains unclear. In this report, NEAT1 has been identified as an important regulator of the host innate immune system against HTNV infection. Elevated NEAT1 promotes IFN secretion, most likely by enhancing RIG-I and DDX60 expression. DDX60, a DEXD/H box RNA helicase similar to Saccharomyces cerevisiae Ski2, is induced after viral infection . . DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response.",
". DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response. However, the antiviral effects of DDX60 seem to vary among viruses . . We found that NEAT1-regulated DDX60 was involved in IFN production in response to HTNV infection. In HTNV-infected cells, double-stranded RNA dsRNA could not be detected, and it is unclear how host PRRs, especially RIG-I, recognize HTNV invasion . . Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation.",
". Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN- production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation. Of note, we applied multiple cell lines to explore the role of NEAT1 during HTNV infection. HTNV primarily targets vascular endothelial cells in vivo and contributes to the increased vascular permeability and coagulation disorders in HFRS; hence, HUVECs are the most common in vitro cell model to study host innate immunity against HTNV infection or viral pathogenesis . . EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies .",
". EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies . . A549 cells were once used to isolate HTNV, and they were confirmed to be a mature model of infection . . . . . Additionally, Huh 7.0 and Huh 7.5 RIG-I Ϫ cells used in our study have been reported to be infected by HTNV by Lee et al. . and can be used as a cell model to study immune responses against HTNV replication . . Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV.",
". Additionally, HEK293 . and HeLa . cells have been reported to be infected by HTNV. Using qRT-PCR, Western blotting, and immunofluorescence assays, we have also shown that both HEK293 and HeLa cells can be infected by HTNV. To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production.",
"To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western ICW analysis was applied to qualify HTNV NP production. Alterations in the relative fluorescence intensity of NP after silencing or overexpressing NEAT1-2 did not seem to be as remarkable as qRT-PCR or Western blot analysis results. The NP spotted and exhibited in the ICW results forms obvious stains that mimic PFU. However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays.",
"However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays. The RNAi studies in vivo are encouraging Fig. 8 , but the NC used by our group was not mutated si-NEAT1-2 i.e., same sense strand, but with a point mutation in the targeting strand . The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling.",
"The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling. After observing that NEAT1 can regulate IFN expression by HTNV infection, we were interested in the function of NEAT1. We noticed that silencing NEAT1-2 or ectopically expressing NEAT1-2 could not inhibit or enhance IFN expression without HTNV infection Fig. 3F , which indicated that NEAT1-2 could not directly affect IFN- expression. This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression.",
"This finding excludes the possibility that NEAT1-2 directly promoted IFN- and that IFN- promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression. Recent findings have shown that RIG-I signaling is essential for an efficient polyfunctional T cell response during IAV infection . . Indeed, we found that the function of T cells was suppressed after NEAT1-2 depletion in our animal experiments Fig. 8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection.",
"8G , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection. HTNV infection induced NEAT1 expression through the RIG-I-IRF7 pathway, while NEAT1 displayed positive feedback for RIG-I signaling. NEAT1 relocated SFPQ from the potential promoter region of several antiviral genes to the paraspeckles, removing the transcriptional inhibitory effects of SFPQ. This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 .",
"This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection Fig. 9 . nontarget control i.e., negative control NC or targeted RIG-I and DDX60 were designed by Gene-Pharma as follows: NC, 5=- UUCUUCGAACGUGUCACGUTT-3=; si-RIG-I-1, 5=-GCCCAUUUAAACCAAGAAATT-3=, si-RIG-I-2, 5=-GGUGGAGGAUAUUUGAACUTT-3=, and si-RIG-I-3, 5=-CCCAACGAUAUCAUUUCUTT-3=; si-DDX60-1, 5=-GUCCAGGUGUCAGUUUGAUTT-3=, si-DDX60-2, 5=-CCGAAGUGAAGAAGGUAAATT-3=, and si-DDX60-3, 5=-GAUGGAUGCUAGGAAAUAUTT-3=. The pCMV-NEAT1-1 and pCMV-NEAT1-2 plasmids, which transcribe NEAT1-1 and NEAT1-2, respectively, were provided by Nakagawa Shinichi . . The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . .",
". The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody MAb 1A8 for the HTNV nucleocapsid protein NP was produced as previously described . . Abs against RIG-I, IRF3, IRF7, p65, and STAT1, as well as the neutralizing antibodies against IFN-␣ and IFN-, were purchased from Abcam Cambridge, MA, USA . Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China .",
"Phorbol myristate acetate PMA and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. St. Louis, MO, USA . Abs against SFPQ, NONO, GAPDH glyceraldehyde-3-phosphate dehydrogenase , and -actin were purchased from Protein Tech, Inc. Wuhan, China . The Abs targeting CD11b, F4/80, CD3, CD8, IFN-␥, inducible nitric oxide synthase iNOS , and CD206 for flow cytometry were purchased from BD Biosciences San Jose, CA, USA . IFN-␣, -, and -␥, TNF-␣, and IL-1 were from PeperoTech Rocky Hill, NJ . ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis.",
"ELISA kits for IFN- detection were manufactured by R&D Systems, Inc. Minneapolis, MN, USA . RNA extraction and quantitative real-time PCR qRT-PCR analysis. Total cellular RNAs were extracted with RNAiso TaKaRa, Dalian, China , the concentration of which was measured using a NanoDrop 1000 spectrophotometer. Reverse transcription RT was then performed with PrimeScript RT master mix TaKaRa according to the instructions provided by the manufacturer. Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed.",
"Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 Roche, Basel, Switzerland . The mRNA expression level of each target gene was normalized to the respective -actin and analyzed. The qRT-PCR primer sequences for NEAT1, NEAT1-2, IFN-, HTNV S segment, RIG-I, DDX60, -actin, and GAPDH were obtained from previous reports . . The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing.",
". The methods used to quantify HTNV RNA load have been described by our group previously . . DGE analysis and lncRNA sequencing. HUVECs with a confluence of 80% in 6 wells were mock infected or infected with live or 60 Co-inactivated HTNV at an MOI of 1. RNAs were extracted as previously described at 24 hpi, and the quality was analyzed using FastQC software by the Beijing Genomics Institute BGI, Shenzhen, China . Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded.",
"Digital gene expression DGE tags were annotated to the human transcriptome Ensembl version 58 by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded. Tag counts were normalized to TPM transcripts per million by dividing the raw tag count by the total number of tags from each library and multiplying by 1 million. To avoid the possible noise signal from high-throughput sequencing, the genes with average TPM of less than 1 in these three states were excluded. In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01.",
"In this study, an absolute fold change of no less than 1.5 and a false discovery rate FDR of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01. Genes were selected as differentially expressed using a P value threshold of 0.01. FISH and immunofluorescence assays IFA . Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature.",
"Fluorescence in situ hybridization FISH was performed with a FISH kit Ribobio Co. according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde PFA for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature. Prehybridization was performed with lncRNA FISH probe mix at 37°C for 30 min, and then hybridization was performed by adding NEAT1-2 FISH probe mix and incubating the mixture at 37°C overnight. After washing with 4ϫ, 2ϫ, and 1ϫ SSC, the cell nuclei were stained with DAPI 4=,6-diamidino-2-phenylindole . Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently.",
"Finally, the samples were observed using a BX60 fluorescence microscope Olympus, Tokyo, Japan . IFA was performed after FISH or independently. The cells were fixed with 4% PFA for 10 min and permeabilized with 0.1% Triton X-100 for 15 min. Primary Abs were added and incubated at 37°C for 2 h. After five washes with DPBS, secondary Cy3-or fluorescein isothiocyanate FITC -conjugated goat anti-rabbit or goat anti-mouse IgG Sangon, Shanghai, China was added and incubated at 37°C for 2 h. Cell nuclei were stained with DAPI. Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue .",
"Finally, the samples were observed using a BX60 fluorescence microscope Olympus . Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer 50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue . The samples were then boiled at 95°C for 10 min. The lysates were resolved by 10%, 12%, or 15% SDS-PAGE and transferred to polyvinylidene fluoride PVDF membranes Millipore . The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA .",
"The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes Li-Cor Biosciences, Lincoln, NE, USA . The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system Li-Cor Biosciences, Lincoln, NE, USA . ICW assay. The In-Cell Western ICW assay was performed using an Odyssey imaging system Li-Cor according to the manufacturer's instructions. HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1.",
"HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates 2 ϫ 10 4 cells/well . Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1. At 48 hpi, HUVECs were washed twice with ice-cold DPBS, fixed with 4% PFA for 10 min, and permeabilized with 1.0% Triton X-100 for 15 min. Cells were added with Li-Cor Odyssey blocking solution at room temperature for 30 min and incubated at 4°C overnight with mouse IgG MAb 1A8 against HTNV NP together with rabbit IgG antibody against -actin, both of which were diluted in PBS containing 3% bovine serum albumin BSA; HyClone . Subsequently, the cells were washed and stained with goat anti-mouse IgG IRDye 800 antibody 1:5,000; Li-Cor and goat anti-rabbit"
] | 2,652 | 5,313 |
What serious question was raised? | as to whether or not our seasonal or pandemic flu might have another reservoir host. | [
"nan Text: pecially with next-generation sequencing NGS for new virus genome discovery, e.g., Ruben Donis et al. sequenced a bat-derived influenza virus genome by using NGS in 2012, raising a serious question as to whether or not our seasonal or pandemic flu might have another reservoir host. Chen and colleagues confirmed the SFTSV independently by using NGS. Indeed, metagenomics analysis has yielded a great deal of new viruses, especially from the environment. Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal .",
"Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal . In this issue, six groups have been invited to present their recent findings on the emerging viruses, in addition to a previous report on H7N9 . Shi reviewed recent discoveries of new viruses or virus genomes from bat. Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 .",
"Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 . Recent MERS-CoV infection is another example for severe disease caused by used-to-be-less pathogenic coronaviruses. Shi and colleagues by using NGS have discovered many unknown animal viruses from bat, especially some important paramyxoviruses and reoviruses. Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city .",
"Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city . The potential roles of these viruses in bats for interspecies transmission are yet to be elucidated. Tan and colleagues specifically focused on the newly-emerged MERS-CoV. The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries.",
"The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries. In the initial diagnosis, the pan-coronavirus real-time reverse transcription polymerase chain reaction RT-PCR assay played a very important role for the identification of the causative agents. By using this method, scientists detected an expected-size PCR fragment for the corresponding conserved region of ORF1b of the replicase gene of a coronavirus. This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently .",
"This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently . Enterovirus has been known as serious human pathogens for a long time but their significance to the public health has been emphasized by the emergence of enterovirus 71 in 1998 as a serious pathogenic agents for children in Taiwan and re-emerged in mainland China in 2008 . In this issue, Duan and colleagues summarized the findings of new enteroviruses by using NGS. Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses.",
"Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses. The review also provided a detailed description of the NGS and related molecular methods for the virus discovery followed by a list of new enteroviruses found in human feces. These include viruses in the family of Piconaviridae, Parvoviridae, Circoviridae, Astroviridae and Polyomaviridae. Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future.",
"Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future. The disease caused by SFTSV, with a CFR of 12%, had been in China for a couple of years before the causative agent was finally identified. There are still a lot of questions remained unknown for this new virus and vigorous studies are in great need. The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts.",
"The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts. Therefore the effective control measures are still under evaluation. Vaccines protecting the SFTSV infection are under its way in Chinese Center for Disease Control and Prevention. Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered.",
"Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered. The new flavivirus, duck egg-drop syndrome virus DEDSV , is a good example. Su and colleagues reviewed the characterization of the DEDSV and its disease form in this issue. The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese .",
"The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese . Yet the transmission vector, though mosquitoes are suspected, has not been identified. Due to the public health concerns of its related viruses, potential human infection of DEDSV should be evaluated. Research on insect viruses is reviving in recent years. In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses.",
"In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses. Studies on these viruses can provide useful knowledge for our understanding about animal or human infecting viruses. More importantly, modification and application of insect-infecting viruses can be used as effective biologicals for the control of insect pest. The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing.",
"The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing. Human beings encounter more ecology-climate-changing problems, including the zoonotic pathogens. We have to face some unknown pathogenic agents passively. To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown.",
"To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown. This is especially true for infectious agents."
] | 2,628 | 3,847 |
What is a recent discovery? | Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host | [
"nan Text: pecially with next-generation sequencing NGS for new virus genome discovery, e.g., Ruben Donis et al. sequenced a bat-derived influenza virus genome by using NGS in 2012, raising a serious question as to whether or not our seasonal or pandemic flu might have another reservoir host. Chen and colleagues confirmed the SFTSV independently by using NGS. Indeed, metagenomics analysis has yielded a great deal of new viruses, especially from the environment. Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal .",
"Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal . In this issue, six groups have been invited to present their recent findings on the emerging viruses, in addition to a previous report on H7N9 . Shi reviewed recent discoveries of new viruses or virus genomes from bat. Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 .",
"Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 . Recent MERS-CoV infection is another example for severe disease caused by used-to-be-less pathogenic coronaviruses. Shi and colleagues by using NGS have discovered many unknown animal viruses from bat, especially some important paramyxoviruses and reoviruses. Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city .",
"Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city . The potential roles of these viruses in bats for interspecies transmission are yet to be elucidated. Tan and colleagues specifically focused on the newly-emerged MERS-CoV. The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries.",
"The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries. In the initial diagnosis, the pan-coronavirus real-time reverse transcription polymerase chain reaction RT-PCR assay played a very important role for the identification of the causative agents. By using this method, scientists detected an expected-size PCR fragment for the corresponding conserved region of ORF1b of the replicase gene of a coronavirus. This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently .",
"This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently . Enterovirus has been known as serious human pathogens for a long time but their significance to the public health has been emphasized by the emergence of enterovirus 71 in 1998 as a serious pathogenic agents for children in Taiwan and re-emerged in mainland China in 2008 . In this issue, Duan and colleagues summarized the findings of new enteroviruses by using NGS. Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses.",
"Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses. The review also provided a detailed description of the NGS and related molecular methods for the virus discovery followed by a list of new enteroviruses found in human feces. These include viruses in the family of Piconaviridae, Parvoviridae, Circoviridae, Astroviridae and Polyomaviridae. Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future.",
"Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future. The disease caused by SFTSV, with a CFR of 12%, had been in China for a couple of years before the causative agent was finally identified. There are still a lot of questions remained unknown for this new virus and vigorous studies are in great need. The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts.",
"The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts. Therefore the effective control measures are still under evaluation. Vaccines protecting the SFTSV infection are under its way in Chinese Center for Disease Control and Prevention. Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered.",
"Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered. The new flavivirus, duck egg-drop syndrome virus DEDSV , is a good example. Su and colleagues reviewed the characterization of the DEDSV and its disease form in this issue. The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese .",
"The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese . Yet the transmission vector, though mosquitoes are suspected, has not been identified. Due to the public health concerns of its related viruses, potential human infection of DEDSV should be evaluated. Research on insect viruses is reviving in recent years. In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses.",
"In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses. Studies on these viruses can provide useful knowledge for our understanding about animal or human infecting viruses. More importantly, modification and application of insect-infecting viruses can be used as effective biologicals for the control of insect pest. The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing.",
"The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing. Human beings encounter more ecology-climate-changing problems, including the zoonotic pathogens. We have to face some unknown pathogenic agents passively. To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown.",
"To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown. This is especially true for infectious agents."
] | 2,628 | 3,848 |
Which bat virus have been found to be linked with diseases? | potential human infecting HKU-1, 4, 5 and 9 . Recent MERS-CoV infection is another example for severe disease caused by used-to-be-less pathogenic coronaviruses. Shi and colleagues by using NGS have discovered many unknown animal viruses from bat, especially some important paramyxoviruses and reoviruses. Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses (with many genotypes, including rabies virus) in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city . | [
"nan Text: pecially with next-generation sequencing NGS for new virus genome discovery, e.g., Ruben Donis et al. sequenced a bat-derived influenza virus genome by using NGS in 2012, raising a serious question as to whether or not our seasonal or pandemic flu might have another reservoir host. Chen and colleagues confirmed the SFTSV independently by using NGS. Indeed, metagenomics analysis has yielded a great deal of new viruses, especially from the environment. Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal .",
"Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal . In this issue, six groups have been invited to present their recent findings on the emerging viruses, in addition to a previous report on H7N9 . Shi reviewed recent discoveries of new viruses or virus genomes from bat. Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 .",
"Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 . Recent MERS-CoV infection is another example for severe disease caused by used-to-be-less pathogenic coronaviruses. Shi and colleagues by using NGS have discovered many unknown animal viruses from bat, especially some important paramyxoviruses and reoviruses. Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city .",
"Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city . The potential roles of these viruses in bats for interspecies transmission are yet to be elucidated. Tan and colleagues specifically focused on the newly-emerged MERS-CoV. The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries.",
"The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries. In the initial diagnosis, the pan-coronavirus real-time reverse transcription polymerase chain reaction RT-PCR assay played a very important role for the identification of the causative agents. By using this method, scientists detected an expected-size PCR fragment for the corresponding conserved region of ORF1b of the replicase gene of a coronavirus. This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently .",
"This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently . Enterovirus has been known as serious human pathogens for a long time but their significance to the public health has been emphasized by the emergence of enterovirus 71 in 1998 as a serious pathogenic agents for children in Taiwan and re-emerged in mainland China in 2008 . In this issue, Duan and colleagues summarized the findings of new enteroviruses by using NGS. Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses.",
"Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses. The review also provided a detailed description of the NGS and related molecular methods for the virus discovery followed by a list of new enteroviruses found in human feces. These include viruses in the family of Piconaviridae, Parvoviridae, Circoviridae, Astroviridae and Polyomaviridae. Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future.",
"Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future. The disease caused by SFTSV, with a CFR of 12%, had been in China for a couple of years before the causative agent was finally identified. There are still a lot of questions remained unknown for this new virus and vigorous studies are in great need. The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts.",
"The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts. Therefore the effective control measures are still under evaluation. Vaccines protecting the SFTSV infection are under its way in Chinese Center for Disease Control and Prevention. Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered.",
"Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered. The new flavivirus, duck egg-drop syndrome virus DEDSV , is a good example. Su and colleagues reviewed the characterization of the DEDSV and its disease form in this issue. The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese .",
"The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese . Yet the transmission vector, though mosquitoes are suspected, has not been identified. Due to the public health concerns of its related viruses, potential human infection of DEDSV should be evaluated. Research on insect viruses is reviving in recent years. In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses.",
"In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses. Studies on these viruses can provide useful knowledge for our understanding about animal or human infecting viruses. More importantly, modification and application of insect-infecting viruses can be used as effective biologicals for the control of insect pest. The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing.",
"The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing. Human beings encounter more ecology-climate-changing problems, including the zoonotic pathogens. We have to face some unknown pathogenic agents passively. To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown.",
"To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown. This is especially true for infectious agents."
] | 2,628 | 3,849 |
What assay played an important role? | reverse transcription polymerase chain reaction (RT-PCR) | [
"nan Text: pecially with next-generation sequencing NGS for new virus genome discovery, e.g., Ruben Donis et al. sequenced a bat-derived influenza virus genome by using NGS in 2012, raising a serious question as to whether or not our seasonal or pandemic flu might have another reservoir host. Chen and colleagues confirmed the SFTSV independently by using NGS. Indeed, metagenomics analysis has yielded a great deal of new viruses, especially from the environment. Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal .",
"Our actively hunting for new viruses has made some significant contributions for our understanding of virus ecology, pathogenesis and interspecies transmission. Science China Life Sciences has focused on this hot topic in the event of the H7N9 outbreak after a comprehensive overview of the topic addressing HPAIV H5N1 in 2009 in the journal . In this issue, six groups have been invited to present their recent findings on the emerging viruses, in addition to a previous report on H7N9 . Shi reviewed recent discoveries of new viruses or virus genomes from bat. Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 .",
"Bat is believed to harbor many more viruses than we ever thought as a reservoir host or even a susceptible host . After the SARS-CoV virus, we have been actively seeking for new coronaviruses from bat and have yielded many of them, including potential human infecting HKU-1, 4, 5 and 9 . Recent MERS-CoV infection is another example for severe disease caused by used-to-be-less pathogenic coronaviruses. Shi and colleagues by using NGS have discovered many unknown animal viruses from bat, especially some important paramyxoviruses and reoviruses. Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city .",
"Filovirus has also been identified in bat with potential severe outcomes. Lyssaviruses with many genotypes, including rabies virus in the Rhabdoviridae family have been linked with severe fatal human cases, even in the developed countries, including Australia, with the bites of bats in the city . The potential roles of these viruses in bats for interspecies transmission are yet to be elucidated. Tan and colleagues specifically focused on the newly-emerged MERS-CoV. The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries.",
"The virus was identified in 2012 in the Middle East with some exported cases to Europe. In 2013 the virus has been re-emerging and expanding its borders to more European countries. In the initial diagnosis, the pan-coronavirus real-time reverse transcription polymerase chain reaction RT-PCR assay played a very important role for the identification of the causative agents. By using this method, scientists detected an expected-size PCR fragment for the corresponding conserved region of ORF1b of the replicase gene of a coronavirus. This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently .",
"This is another example that molecular biology methods played for the discovery of new pathogens. Soon the receptor used by MERS-CoV to enter the host cells was identified and the molecular basis of the receptor binding to the virus was also elucidated recently . Enterovirus has been known as serious human pathogens for a long time but their significance to the public health has been emphasized by the emergence of enterovirus 71 in 1998 as a serious pathogenic agents for children in Taiwan and re-emerged in mainland China in 2008 . In this issue, Duan and colleagues summarized the findings of new enteroviruses by using NGS. Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses.",
"Because of the application of new NGS technology they also challenged the Koch's postulates. A new model of Koch's postulates, named the metagenomic Koch's postulates, has provided guidance for the study of the pathogenicity of novel viruses. The review also provided a detailed description of the NGS and related molecular methods for the virus discovery followed by a list of new enteroviruses found in human feces. These include viruses in the family of Piconaviridae, Parvoviridae, Circoviridae, Astroviridae and Polyomaviridae. Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future.",
"Yu Xue-Jie and colleagues reviewed the new bunyavirus, SFTSV, identified in China. As the virus discoverers, they have overviewed the whole process of the discovery, which is helpful and meaningful for the new virus discoveries in the future. The disease caused by SFTSV, with a CFR of 12%, had been in China for a couple of years before the causative agent was finally identified. There are still a lot of questions remained unknown for this new virus and vigorous studies are in great need. The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts.",
"The transmission route of the virus has not been clarified but tick as vector is suspected. Domestic and wild animals, e.g., goats, boars, cattle and dogs, are believed to be the virus-amplifying hosts. Therefore the effective control measures are still under evaluation. Vaccines protecting the SFTSV infection are under its way in Chinese Center for Disease Control and Prevention. Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered.",
"Recently a similar virus has been identified in both Japan and USA a new name of Heartland virus was proposed for the US virus . In addition to new viruses infecting human beings, some new viruses infecting animals but their public health significance needing to be further evaluated, have also been discovered. The new flavivirus, duck egg-drop syndrome virus DEDSV , is a good example. Su and colleagues reviewed the characterization of the DEDSV and its disease form in this issue. The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese .",
"The virus was found closely-related to a long-time-known virus, Tembusu virus . Initially, the disease was only found in egg-raising ducks but soon it was found in pigeons, chickens and geese . Yet the transmission vector, though mosquitoes are suspected, has not been identified. Due to the public health concerns of its related viruses, potential human infection of DEDSV should be evaluated. Research on insect viruses is reviving in recent years. In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses.",
"In this issue, Zhou and colleagues reviewed the newly-identified insect viruses in China. Insects are the largest group of animals on the Earth therefore they also carry many more viruses. Studies on these viruses can provide useful knowledge for our understanding about animal or human infecting viruses. More importantly, modification and application of insect-infecting viruses can be used as effective biologicals for the control of insect pest. The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing.",
"The new viruses identified include Wuhan nodavirus WhNV , a member of family Nodaviridae; Dendrolimus punctatus tetravirus DpTV , a new member of the genus Omegatetravirus of the family Alphatetravirida; Ectropis obliqua picorna-like virus EoV , a positive-strand RNA virus causing a lethal granulosis infection in the larvae of the tea looper Ectropis obliqua , the virus a member of the Flaviridae family. While we are enjoying ourselves with the civilization of modern societies, the ecology has ever been changing. Human beings encounter more ecology-climate-changing problems, including the zoonotic pathogens. We have to face some unknown pathogenic agents passively. To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown.",
"To get ourselves well prepared we also ought to actively hunt for unknown pathogens. Prediction and pre-warning can only be realized by knowing more about the unknown. This is especially true for infectious agents."
] | 2,628 | 3,850 |
What was the death toll in the 1918-1919 Spanish Influenza epidemic? | 50 million deaths worldwide | [
"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.",
"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..",
"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.",
"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.",
"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.",
"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .",
"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.",
"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .",
"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.",
"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .",
"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.",
"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .",
"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.",
"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.",
"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.",
"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.",
"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.",
"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.",
"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?",
"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.",
"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.",
"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.",
"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.",
"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.",
"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.",
"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?",
"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?",
"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.",
"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..",
"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.",
"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.",
"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.",
"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.",
"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .",
"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.",
"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.",
"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.",
"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.",
"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.",
"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.",
"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.",
"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.",
"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.",
"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.",
"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.",
"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.",
"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.",
"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.",
"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.",
"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.",
"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.",
"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.",
"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.",
"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.",
"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.",
"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.",
"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.",
"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.",
"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.",
"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."
] | 2,684 | 1,057 |
How many people were infected during the 1918 Spanish Influenza epidemic? | An estimated one third of the world’s population (or
z500 million persons) were infected and had clinical-
ly apparent illnesses | [
"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.",
"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..",
"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.",
"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.",
"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.",
"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .",
"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.",
"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .",
"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.",
"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .",
"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.",
"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .",
"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.",
"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.",
"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.",
"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.",
"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.",
"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.",
"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?",
"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.",
"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.",
"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.",
"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.",
"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.",
"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.",
"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?",
"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?",
"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.",
"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..",
"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.",
"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.",
"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.",
"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.",
"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .",
"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.",
"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.",
"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.",
"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.",
"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.",
"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.",
"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.",
"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.",
"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.",
"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.",
"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.",
"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.",
"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.",
"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.",
"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.",
"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.",
"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.",
"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.",
"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.",
"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.",
"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.",
"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.",
"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.",
"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.",
"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.",
"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."
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What was the case fatality rate in the 1918 Spanish Influenza epidemic? | Case-
fatality rates were >2.5%, compared to <0.1% in other
influenza pandemics | [
"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.",
"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..",
"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.",
"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.",
"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.",
"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .",
"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.",
"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .",
"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.",
"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .",
"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.",
"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .",
"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.",
"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.",
"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.",
"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.",
"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.",
"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.",
"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?",
"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.",
"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.",
"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.",
"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.",
"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.",
"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.",
"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?",
"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?",
"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.",
"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..",
"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.",
"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.",
"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.",
"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.",
"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .",
"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.",
"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.",
"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.",
"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.",
"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.",
"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.",
"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.",
"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.",
"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.",
"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.",
"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.",
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"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.",
"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.",
"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.",
"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.",
"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.",
"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.",
"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.",
"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."
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What was the death toll in the 1918-1919 Spanish Influenza epidemic? | Total deaths were estimated at
z50 million (577) and were arguably as high as 100 mil-
lion | [
"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.",
"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..",
"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.",
"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.",
"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.",
"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .",
"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.",
"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .",
"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.",
"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .",
"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.",
"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .",
"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.",
"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.",
"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.",
"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.",
"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.",
"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.",
"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?",
"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.",
"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.",
"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.",
"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.",
"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.",
"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.",
"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?",
"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?",
"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.",
"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..",
"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.",
"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.",
"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.",
"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.",
"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .",
"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.",
"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.",
"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.",
"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.",
"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.",
"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.",
"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.",
"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.",
"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.",
"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.",
"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.",
"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.",
"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.",
"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.",
"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.",
"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.",
"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.",
"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.",
"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.",
"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.",
"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.",
"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.",
"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.",
"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.",
"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.",
"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."
] | 2,684 | 1,060 |
Are the modern day Influenza viruses related to the 1918 Spanish Influenza virus? | All influenza A pandemics since that time, and
indeed almost all cases of influenza A worldwide (except-
ing human infections from avian Viruses such as H5N1 and
H7N7), have been caused by descendants of the 1918
Virus, including “drifted” H1N1 Viruses and reassorted
H2N2 and H3N2 Viruses. | [
"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.",
"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..",
"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.",
"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.",
"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.",
"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .",
"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.",
"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .",
"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.",
"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .",
"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.",
"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .",
"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.",
"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.",
"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.",
"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.",
"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.",
"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.",
"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?",
"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.",
"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.",
"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.",
"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.",
"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.",
"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.",
"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?",
"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?",
"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.",
"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..",
"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.",
"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.",
"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.",
"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.",
"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .",
"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.",
"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.",
"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.",
"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.",
"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.",
"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.",
"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.",
"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.",
"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.",
"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.",
"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.",
"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.",
"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.",
"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.",
"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.",
"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.",
"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.",
"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.",
"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.",
"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.",
"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.",
"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.",
"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.",
"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.",
"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.",
"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."
] | 2,684 | 1,062 |
Why is the Spanish Influenza virus the Mother of the modern influenza viruses? | The latter are composed of key
genes from the 1918 Virus, updated by subsequently-incor—
porated avian influenza genes that code for novel surface
*Armed Forces Institute of Pathology, Rockville, Maryland, USA;
and TNational Institutes of Health, Bethesda, Maryland, USA
proteins, making the 1918 Virus indeed the “mother” of all
pandemics. | [
"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.",
"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..",
"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.",
"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.",
"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.",
"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .",
"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.",
"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .",
"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.",
"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .",
"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.",
"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .",
"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.",
"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.",
"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.",
"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.",
"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.",
"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.",
"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?",
"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.",
"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.",
"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.",
"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.",
"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.",
"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.",
"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?",
"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?",
"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.",
"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..",
"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.",
"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.",
"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.",
"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.",
"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .",
"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.",
"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.",
"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.",
"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.",
"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.",
"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.",
"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.",
"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.",
"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.",
"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.",
"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.",
"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.",
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"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.",
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"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.",
"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.",
"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."
] | 2,684 | 1,063 |
When was it determined that the 1918 pandemic was caused by the H1N1 Influenza virus? | That question did not begin to be resolved until the 1930s,
when closely related influenza Viruses (now known to be
H1N1 Viruses) were isolated, first from pigs and shortly
thereafter from humans. Seroepidemiologic studies soon
linked both of these viruses to the 1918 pandemic | [
"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.",
"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..",
"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.",
"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.",
"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.",
"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .",
"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.",
"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .",
"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.",
"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .",
"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.",
"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .",
"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.",
"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.",
"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.",
"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.",
"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.",
"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.",
"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?",
"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.",
"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.",
"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.",
"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.",
"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.",
"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.",
"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?",
"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?",
"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.",
"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..",
"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.",
"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.",
"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.",
"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.",
"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .",
"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.",
"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.",
"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.",
"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.",
"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.",
"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.",
"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.",
"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.",
"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.",
"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.",
"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.",
"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.",
"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.",
"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.",
"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.",
"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.",
"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.",
"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.",
"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.",
"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.",
"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.",
"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.",
"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.",
"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.",
"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.",
"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."
] | 2,684 | 1,064 |
Did the Spanish Influenza or Swine flu or the H1N1 virus disappear in humans for some time? | descendants of the 1918
Virus still persists enzootically in pigs. They probably also
circulated continuously in humans, undergoing gradual
antigenic drift and causing annual epidemics, until the
1950s. With the appearance of a new H2N2 pandemic
strain in 1957 (“Asian flu”), the direct H1N1 Viral descen-
dants 0f the 1918 pandemic strain disappeared from human
circulation entirely, although the related lineage persisted
enzootically in pigs. | [
"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.",
"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..",
"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.",
"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.",
"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.",
"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .",
"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.",
"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .",
"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.",
"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .",
"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.",
"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .",
"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.",
"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.",
"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.",
"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.",
"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.",
"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.",
"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?",
"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.",
"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.",
"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.",
"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.",
"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.",
"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.",
"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?",
"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?",
"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.",
"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..",
"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.",
"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.",
"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.",
"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.",
"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .",
"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.",
"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.",
"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.",
"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.",
"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.",
"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.",
"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.",
"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.",
"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.",
"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.",
"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.",
"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.",
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"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.",
"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.",
"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.",
"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.",
"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.",
"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.",
"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.",
"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.",
"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.",
"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.",
"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."
] | 2,684 | 1,065 |
When did the Swine Flu (Spanish Influenza) virus reappear in humans? | But in 1977, human H1N1 Viruses
suddenly “reemerged” from a laboratory freezer (9). They
continue to circulate endemically and epidemically. | [
"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.",
"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..",
"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.",
"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.",
"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.",
"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .",
"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.",
"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .",
"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.",
"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .",
"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.",
"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .",
"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.",
"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.",
"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.",
"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.",
"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.",
"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.",
"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?",
"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.",
"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.",
"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.",
"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.",
"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.",
"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.",
"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?",
"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?",
"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.",
"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..",
"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.",
"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.",
"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.",
"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.",
"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .",
"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.",
"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.",
"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.",
"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.",
"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.",
"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.",
"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.",
"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.",
"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.",
"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.",
"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.",
"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.",
"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.",
"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.",
"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.",
"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.",
"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.",
"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.",
"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.",
"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.",
"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.",
"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.",
"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.",
"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.",
"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.",
"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."
] | 2,684 | 1,066 |
What descendant lineages of the swine flu (Spanish Influenza) virus were identified in 2006? | 2 major descendant lineages of the 1918
H1N1 Virus, as well as 2 additional reassortant lineages,
persist naturally: a human epidemic/endemic H1N1 line-
age, a porcine enzootic H1N1 lineage (so-called classic
swine flu), and the reassorted human H3N2 Virus lineage,
which like the human H1N1 Virus, has led to a porcine
H3N2 lineage. | [
"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.",
"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..",
"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.",
"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.",
"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.",
"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .",
"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.",
"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .",
"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.",
"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .",
"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.",
"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .",
"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.",
"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.",
"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.",
"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.",
"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.",
"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.",
"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?",
"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.",
"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.",
"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.",
"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.",
"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.",
"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.",
"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?",
"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?",
"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.",
"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..",
"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.",
"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.",
"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.",
"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.",
"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .",
"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.",
"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.",
"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.",
"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.",
"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.",
"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.",
"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.",
"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.",
"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.",
"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.",
"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.",
"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.",
"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.",
"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.",
"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.",
"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.",
"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.",
"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.",
"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.",
"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.",
"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.",
"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.",
"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.",
"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.",
"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.",
"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."
] | 2,684 | 1,068 |
Are the modern descendant influenza viruses as dangerous as the 1918 parent swine flu (Spanish Influenza) H1N1 virus? | None of these Viral descendants, however,
approaches the pathogenicity of the 1918 parent Virus. | [
"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.",
"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..",
"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.",
"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.",
"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.",
"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .",
"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.",
"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .",
"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.",
"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .",
"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.",
"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .",
"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.",
"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.",
"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.",
"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.",
"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.",
"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.",
"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?",
"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.",
"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.",
"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.",
"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.",
"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.",
"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.",
"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?",
"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?",
"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.",
"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..",
"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.",
"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.",
"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.",
"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.",
"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .",
"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.",
"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.",
"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.",
"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.",
"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.",
"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.",
"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.",
"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.",
"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.",
"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.",
"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.",
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"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.",
"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.",
"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."
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How dangerous are the modern H1N1 (swine flu) and the H3N2 (Influenza A) viruses compared to the 1918 H1N1 (swine flu Spanish Influenza) viruses? | the human H1N1 and H3N2 lin-
eages have both been associated with substantially lower
rates ofillness and death than the virus of 1918. In fact, cur-
rent H1N1 death rates are even lower than those for H3N2
lineage strains (prevalent from 1968 until the present). | [
"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.",
"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..",
"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.",
"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.",
"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.",
"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .",
"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.",
"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .",
"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.",
"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .",
"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.",
"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .",
"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.",
"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.",
"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.",
"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.",
"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.",
"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.",
"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?",
"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.",
"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.",
"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.",
"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.",
"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.",
"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.",
"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?",
"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?",
"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.",
"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..",
"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.",
"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.",
"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.",
"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.",
"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .",
"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.",
"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.",
"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.",
"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.",
"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.",
"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.",
"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.",
"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.",
"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.",
"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.",
"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.",
"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.",
"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.",
"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.",
"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.",
"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.",
"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.",
"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.",
"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.",
"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.",
"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.",
"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.",
"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.",
"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.",
"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.",
"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."
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Are the descendant H1N1 strains of the 1918 H1N1 swine flu (Spanish Influenza) virus, still prevalent? | H1N1 Viruses descended from the 1918 strain, as well as
H3N2 Viruses, have now been cocirculating worldwide for
29 years and show little evidence of imminent extinction. | [
"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.",
"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..",
"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.",
"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.",
"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.",
"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .",
"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.",
"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .",
"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.",
"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .",
"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.",
"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .",
"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.",
"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.",
"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.",
"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.",
"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.",
"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.",
"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?",
"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.",
"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.",
"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.",
"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.",
"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.",
"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.",
"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?",
"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?",
"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.",
"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..",
"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.",
"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.",
"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.",
"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.",
"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .",
"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.",
"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.",
"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.",
"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.",
"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.",
"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.",
"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.",
"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.",
"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.",
"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.",
"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.",
"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.",
"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.",
"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.",
"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.",
"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.",
"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.",
"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.",
"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.",
"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.",
"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.",
"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.",
"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.",
"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.",
"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.",
"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."
] | 2,684 | 1,073 |
Is the origin and epidemiology of the 1918 swine flu (Spanish Influenza) known? | ongoing studies to map Virulence
factors are yielding interesting results. The 1918 sequence
data, however, leave unanswered questions about the ori-
gin of the Virus (19) and about the epidemiology of the
pandemic. | [
"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.",
"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..",
"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.",
"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.",
"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.",
"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .",
"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.",
"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .",
"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.",
"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .",
"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.",
"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .",
"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.",
"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.",
"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.",
"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.",
"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.",
"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.",
"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?",
"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.",
"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.",
"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.",
"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.",
"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.",
"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.",
"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?",
"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?",
"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.",
"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..",
"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.",
"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.",
"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.",
"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.",
"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .",
"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.",
"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.",
"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.",
"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.",
"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.",
"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.",
"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.",
"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.",
"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.",
"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.",
"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.",
"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.",
"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.",
"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.",
"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.",
"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.",
"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.",
"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.",
"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.",
"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.",
"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.",
"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.",
"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.",
"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.",
"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.",
"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."
] | 2,684 | 1,075 |
What is the geographical origin of the H1N1 swine flu ? | Historical and epidemiologic data are inade-
quate to identify the geographic origin of the Virus (21),
and recent phylogenetic analysis of the 1918 Viral genome
does not place the Virus in any geographic context | [
"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.",
"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..",
"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.",
"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.",
"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.",
"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .",
"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.",
"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .",
"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.",
"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .",
"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.",
"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .",
"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.",
"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.",
"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.",
"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.",
"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.",
"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.",
"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?",
"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.",
"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.",
"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.",
"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.",
"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.",
"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.",
"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?",
"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?",
"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.",
"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..",
"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.",
"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.",
"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.",
"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.",
"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .",
"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.",
"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.",
"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.",
"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.",
"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.",
"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.",
"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.",
"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.",
"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.",
"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.",
"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.",
"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.",
"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.",
"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.",
"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.",
"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.",
"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.",
"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.",
"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.",
"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.",
"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.",
"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.",
"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.",
"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.",
"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.",
"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."
] | 2,684 | 1,082 |
Is the geographical origin of the 1918 H1N1 swine flu known? | Confounding definite assignment of a geographic
point of origin, the 1918 pandemic spread more or less
simultaneously in 3 distinct waves during an z12-month
period in 191871919, in Europe, Asia, and North America
(the first wave was best described in the United States in
March 1918) | [
"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.",
"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..",
"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.",
"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.",
"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.",
"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .",
"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.",
"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .",
"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.",
"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .",
"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.",
"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .",
"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.",
"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.",
"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.",
"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.",
"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.",
"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.",
"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?",
"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.",
"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.",
"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.",
"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.",
"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.",
"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.",
"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?",
"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?",
"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.",
"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..",
"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.",
"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.",
"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.",
"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.",
"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .",
"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.",
"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.",
"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.",
"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.",
"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.",
"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.",
"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.",
"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.",
"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.",
"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.",
"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.",
"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.",
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"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.",
"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.",
"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.",
"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.",
"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.",
"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.",
"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.",
"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."
] | 2,684 | 1,087 |
What is an unique feature of the 1918 swine flu? | the simultaneous (or nearly simultaneous) infection
of humans and swin | [
"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.",
"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..",
"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.",
"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.",
"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.",
"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .",
"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.",
"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .",
"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.",
"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .",
"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.",
"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .",
"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.",
"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.",
"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.",
"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.",
"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.",
"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.",
"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?",
"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.",
"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.",
"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.",
"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.",
"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.",
"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.",
"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?",
"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?",
"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.",
"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..",
"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.",
"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.",
"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.",
"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.",
"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .",
"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.",
"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.",
"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.",
"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.",
"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.",
"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.",
"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.",
"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.",
"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.",
"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.",
"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.",
"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.",
"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.",
"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.",
"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.",
"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.",
"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.",
"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.",
"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.",
"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.",
"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.",
"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.",
"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.",
"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.",
"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.",
"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."
] | 2,684 | 1,088 |
What season or time of the year do the new strains of influenza emerge? | Historical records since the 16th century suggest that
new influenza pandemics may appear at any time of year,
not necessarily in the familiar annual winter patterns of
interpandemic years, | [
"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.",
"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..",
"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.",
"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.",
"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.",
"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .",
"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.",
"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .",
"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.",
"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .",
"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.",
"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .",
"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.",
"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.",
"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.",
"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.",
"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.",
"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.",
"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?",
"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.",
"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.",
"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.",
"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.",
"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.",
"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.",
"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?",
"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?",
"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.",
"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..",
"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.",
"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.",
"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.",
"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.",
"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .",
"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.",
"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.",
"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.",
"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.",
"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.",
"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.",
"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.",
"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.",
"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.",
"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.",
"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.",
"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.",
"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.",
"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.",
"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.",
"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.",
"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.",
"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.",
"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.",
"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.",
"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.",
"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.",
"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.",
"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.",
"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.",
"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."
] | 2,684 | 1,089 |
Once appeared, when do the influenza like diseases occur in subsequent years? | confronted by the selection pressures of population immu-
nity, these pandemic Viruses begin to drift genetically and
eventually settle into a pattern of annual epidemic recur-
rences caused by the drifted Virus variants. | [
"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.",
"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..",
"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.",
"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.",
"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.",
"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .",
"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.",
"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .",
"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.",
"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .",
"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.",
"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .",
"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.",
"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.",
"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.",
"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.",
"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.",
"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.",
"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?",
"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.",
"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.",
"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.",
"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.",
"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.",
"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.",
"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?",
"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?",
"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.",
"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..",
"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.",
"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.",
"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.",
"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.",
"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .",
"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.",
"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.",
"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.",
"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.",
"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.",
"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.",
"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.",
"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.",
"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.",
"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.",
"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.",
"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.",
"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.",
"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.",
"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.",
"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.",
"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.",
"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.",
"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.",
"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.",
"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.",
"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.",
"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.",
"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.",
"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.",
"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."
] | 2,684 | 1,090 |
When did the first wave of the H1N1 swine flu (Spanish Influenza) occur? | a first or spring wave
began in March 1918 and spread unevenly through the
United States, Europe, and possibly Asia over the next 6
months | [
"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.",
"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..",
"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.",
"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.",
"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.",
"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .",
"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.",
"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .",
"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.",
"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .",
"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.",
"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .",
"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.",
"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.",
"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.",
"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.",
"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.",
"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.",
"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?",
"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.",
"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.",
"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.",
"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.",
"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.",
"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.",
"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?",
"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?",
"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.",
"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..",
"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.",
"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.",
"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.",
"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.",
"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .",
"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.",
"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.",
"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.",
"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.",
"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.",
"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.",
"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.",
"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.",
"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.",
"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.",
"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.",
"His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.",
"Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.",
"Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.",
"Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.",
"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.",
"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.",
"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.",
"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.",
"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.",
"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.",
"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.",
"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.",
"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.",
"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.",
"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."
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in most locales were not appreciably above normal. | [
"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.",
"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..",
"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.",
"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.",
"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.",
"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .",
"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.",
"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .",
"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.",
"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .",
"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.",
"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .",
"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.",
"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.",
"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.",
"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.",
"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.",
"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.",
"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?",
"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.",
"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.",
"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.",
"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.",
"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.",
"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.",
"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?",
"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?",
"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.",
"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..",
"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.",
"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.",
"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.",
"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.",
"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .",
"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.",
"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.",
"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.",
"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.",
"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.",
"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.",
"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.",
"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.",
"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.",
"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.",
"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.",
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"12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.",
"J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.",
"Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.",
"Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.",
"Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.",
"Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.",
"Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.",
"A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.",
"J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.",
"J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.",
"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."
] | 2,684 | 1,092 |
When were the second and the third wave of the 1918-1919 swine flu pandemic? | A sec-
ond or fall wave spread globally from September to
November 1918 and was highly fatal. In many nations, a
third wave occurred in early 1919 | [
"1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.",
"But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..",
"Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.",
"The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.",
"Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.",
"But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .",
"Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.",
"No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .",
"These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.",
"The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .",
"Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.",
"Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .",
"The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.",
"Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.",
"Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.",
"In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.",
"The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.",
"The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.",
"However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?",
"Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.",
"And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.",
"As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.",
"Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.",
"Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.",
"One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.",
"Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?",
"One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?",
"Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.",
"Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..",
"The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.",
"Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.",
"These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.",
"The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.",
"The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .",
"The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.",
"But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.",
"But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.",
"Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.",
"In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.",
"In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.",
"Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.",
"Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.",
"There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.",
"Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.",
"Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.",
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"Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated."
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